Human papilloma virus as predictor of cancer prognosis

ABSTRACT

Methods of treating a head and neck cancer are disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/200,088, filed Jul. 1, 2016, now allowed, which is a continuation ofU.S. patent application Ser. No. 14/177,615, filed Feb. 11, 2014, nowU.S. Pat. No. 9,410,954, issued Aug. 9, 2016, which is a continuation ofU.S. patent application Ser. No. 13/958,502, filed Aug. 2, 2013, nowU.S. Pat. No. 8,673,972, issued Mar. 18, 2014, which claims the benefitof U.S. Provisional Application No. 61/679,354, filed Aug. 3, 2012, thecontents of which are hereby incorporated by reference in theirentirety.

BACKGROUND

Cancer represents the phenotypic end-point of multiple genetic lesionsthat endow cells with a full range of biological properties required fortumorigenesis. Indeed, a hallmark genomic feature of many cancers,including, for example, cancers of the head and neck, breast cancer,prostate cancer, ovarian cancer, endometrial cancer, and colon cancer,is the presence of numerous complex chromosome structural aberrations,including translocations, intra-chromosomal inversions, point mutations,deletions, gene copy number changes, gene expression level changes, andgermline mutations, among others. Whether a cancer will respond to aparticular treatment option may depend on the particular genomicfeatures present in the cancer.

The need still exists for identifying effective treatment options forcancer. Identification of genetic features in a cancer can be aneffective approach to develop compositions, methods and assays forevaluating and treating the cancer.

SUMMARY

The invention is based, at least in part, on the discovery that subjectswith a cancer, such as a head and neck cancer (e.g., a head and necksquamous cell carcinoma (HNSCC)), have certain genetic alterationsdepending on whether or not they carry the human papillomavirus (HPV).In one embodiment, Applicants have discovered that subjects with a headand neck cancer, who are also HPV-negative (HPV−), are more likely tocarry a genomic alteration (e.g., a copy number alteration or amutation) in a cell cycle gene, such as in one or more of CDKN2A (cyclindependent kinase inhibitor 2A), CDKN2B (cyclin dependent kinaseinhibitor 2B), CCNE1 (Cyclin E1), CCND1 (Cyclin D1), CCND2 (Cyclin D2),CCND3 (Cyclin D3), CDK4 (cyclin dependent kinase 4) or CDK6 (cyclindependent kinase 6). Thus, a subject with an HPV− status can be treatedwith a drug that targets the cell cycle gene, or a gene or protein thatfunctions downstream of the cell cycle gene. For example, a HNSCCsubject with an HPV− status can be treated with a CDK (cyclin dependentkinase) inhibitor, which will target CDK proteins overexpressed due to aCDKN2A or CDKN2B loss-of-function mutation, such as a CDKN2A or CDKN2Bdeletion. Further, genomic profiling (e.g., acquiring the sequence ofall or part of a cyclin-dependent kinase inhibitor, a cyclin, or acyclin-dependent kinase, e.g., CDKN2A, CDKN2B, CCNE1, CCND1, CCND2,CCND3, CDK4 or CDK6), is not necessary before making the determinationthat the HPV−/HNSCC subject is likely to respond to treatment with a CDKinhibitor, or before administering the CDK inhibitor. In otherembodiments, Applicants have discovered that subjects with a head andneck cancer, who are HPV-positive (HPV+), tend to have a higherfrequency of PI3 Kinase (PI3K) alterations (e.g., copy numberalterations or mutations), and a lower frequency of alterations in cellcycle genes. Thus, subjects who are HPV+ are less likely to respond to atreatment with a drug that targets a cell cycle gene, or a gene orprotein that functions downstream of the cell cycle gene. For example, aHNSCC subject with an HPV+ status can be treated with a drug other thana CDK (cyclin dependent kinase) inhibitor, or a CCND1 inhibitor. TheHPV+ HNSCC patient can alternatively, be treated with a PI3K inhibitorand/or an mTOR inhibitor. In one embodiment, HPV+ HNSCC patient issubjected to genomic profiling to confirm abnormal upregulation of PI3Kprior to treatment with the PI3K inhibitor. Thus, evaluation ofHPV-status in a subject with a cancer, e.g., a head and neck cancer, canbe used to evaluate cancer responsiveness. In other embodiments,identification of an alteration (e.g., a mutation) in a cell cycle geneis indicative that the cancer is more responsive to a CDK inhibitor.Therefore, the invention provides methods, assays and kits forevaluating, identifying, assessing, evaluating, and/or treating asubject having a cancer, e.g., a head and neck cancer.

Accordingly, in one aspect, the invention features a method of treatinga subject having a cancer, e.g., a head and neck cancer (e.g., anHNSCC). The method includes:

acquiring knowledge of an HPV-status (e.g., the presence or absence ofHPV) in a subject, and

responsive to the determination that the subject is HPV⁻, administeringto the subject an inhibitor that targets a cell-cycle gene, such as aCDK inhibitor. In one embodiment, the subject is further administeredradiation therapy, or is further administered a surgery to treat thecancer.

In certain embodiments, the method further includes evaluating thesubject for the presence or absence of an alteration (e.g., a mutationor a copy number alteration) in a cell cycle gene, e.g., a CDKN2A gene,a CDKN2B gene, CCNE1 gene, CCND1 gene, CCND2 gene, CCND3 gene, CDK4 geneor CDK6 gene.

In one embodiment, the subject, e.g., an HPV− patient, is administered aCDK inhibitor that inhibits one or both of CDK4 or CDK6 (e.g., aninhibitor that inhibits both CDK4 and CDK6, i.e., a CDK4/6 inhibitor).In one embodiment, the CDK4/6 inhibitor is LEE011 (Novartis),LY-2835219, or PD 0332991 (Pfizer). In one embodiment, the CDK inhibitorinhibits CDK1, CDK2, CDK7, and/or CDK9. In certain embodiments, the CDKinhibitor is flavopiridol, indisulam, AZD5438, SNS-032, SCH 727965(Dinaciclib), JNJ-7706621, indirubin, or seliciclib. In one embodiment,the CDK inhibitor is not flavopiridol.

In one embodiment, the subject is HPV−, and has a mutation in a cellcycle gene, such as the CDKN2A gene or CDKN2B gene, or the CCND1 gene.In another embodiment, the mutation in the CDKN2A gene is aloss-of-function mutation. For example, the mutant CDKN2A gene has adeletion, such as a homozygous deletion, or one or more point mutations.In another embodiment, the mutation in the CCND1 gene is again-of-function mutation. For example, the CCND1 gene can be amplified.In one embodiment, the cell cycle gene has a mutation as described inTable 4.

In certain embodiments, the subject has a localized cancer, e.g., alocalized cancer of the head or neck. In other embodiments, the subjecthas metastatic cancer. In certain embodiments, the subject is furtherevaluated for the presence of one or more of the alterations, e.g.,mutations, disclosed herein. In one embodiment, the treatment of thesubject is modified, e.g., decreased, discontinued, or otherwisealtered, in response to the detection of one or more of the alterations,e.g., mutations, described herein.

In one embodiment, the step of acquiring knowledge of the HPV status inthe method includes detecting or identifying an HPV molecule in thesubject, e.g., in a sample from the subject. In one embodiment, the HPVmolecule is an HPV nucleic acid or HPV protein in the subject, e.g., thesample. In some embodiments, an HPV nucleic acid is identified by insitu hybridization (ISH), PCR, Northern blot analysis, or sequencing ofnucleic acids in the sample. HPV protein can be identified, e.g.,immunohistochemistry (IHC), by Western blot analysis. In one embodiment,HPV is detected by IHC to detect p16 protein (encoded by CDKN2A), whichis strongly and diffusely expressed in about 93% of HPV-associatedsquamous cell carcinomas (SCCs). A sample from a subject can be, forexample, a blood or serum sample, or a urine sample, or a tissue sample,such as a tumor tissue sample (such as from a biopsy), or a buccal swab.The sample can be fresh or frozen.

In one embodiment, determination that the subject is HPV− is sufficientto conclude that the subject is a candidate to receive treatment with aCDK inhibitor, e.g., a CDK4/6 inhibitor. Further genomic profiling isnot necessary before making the determination that the subject is likelyto respond to treatment with a CDK inhibitor, or before administeringthe CDK inhibitor.

In one embodiment, the alteration of the cell cycle gene is detected ina nucleic acid molecule or polypeptide in a biological sample from thesubject. For example, the sample can include a fluid, such as blood,plasma, saliva, or urine; cells, such as from a buccal swab; or atissue, such as a tumor tissue, such as from a biopsy. The biologicalsample can be acquired from a subject, or from a depository, forexample.

In one embodiment, the sample includes tissue or a nucleic acid, such asfrom a tumor biopsy or a circulating tumor cell.

In one embodiment, a mutant cell cycle polypeptide is detected. Forexample, in certain embodiments, the mutant cell cycle polypeptide is amutant CDKN2A polypeptide, a mutant CDKN2B polypeptide, a mutant CCNE1polypeptide, a mutant CCND1 polypeptide, a mutant CCND2 polypeptide, amutant CCND3 polypeptide, a mutant CDK4 polypeptide or a mutant CDK6polypeptide. In one embodiment, a biological sample is positive forCCND1 immunohistochemistry, and the positive staining is greater than ina non-tumor control sample. In one embodiment, a change in levels of aprotein upstream or downstream of a mutant cell cycle gene is detected.For example, detection of increased levels of CDK protein (e.g., a CDK4or CDK6 protein) is indicative of a loss-of-function mutation in aCDKN2A or CDKN2B gene.

In one embodiment, a mutant cell-cycle gene is detected in a nucleicacid molecule, such as a nucleic acid molecule isolated from a tumortissue or a circulating tumor cell. The mutation in the cell-cycle genecan be detected by a method known in the art, such as a nucleic acidhybridization assay, an amplification-based assay, a PCR-RFLP assay,real-time PCR, sequencing, screening analysis, FISH (fluorescence insitu hybridization), spectral karyotyping or MFISH (multicolor FISH),comparative genomic hybridization, in situ hybridization, SSP (sequencespecific primers), HPLC (high performance liquid chromatography) ormass-spectrometric genotyping.

In another embodiment, the level or activity of the mutant cell-cyclegene is evaluated.

A mutation in the cell cycle gene can be detected, for example, prior toinitiating, during, or after, treatment of a subject. In one embodiment,the mutation in the cell cycle gene is detected at the time of diagnosiswith a cancer.

In one embodiment, the subject is HPV⁺, and the subject is administeredan agent (e.g., an anti-cancer agent) other than a CDK (cyclin-dependentkinase) inhibitor, e.g., other than a CDK4/6 inhibitor. For example, theHPV+ patient is administered 4-hydroxyperoxycyclophosphoramide,5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), 5-azacytidine,6-mercaptopurine, 6-thioguanine, actinomycin D, amsacrine,bis-chloroethylnitrosurea, bleomycin, bryostatin-1, busulfan,carboplatin (Paraplatin®), chlorambucil, cisplatin (Platinol®),cetuximab (Erbitux®), colchicine, cyclophosphamide, cytarabine, cytosinearabinoside, dacarbazine, daunorubicin, daunomycin, dactinomycin,deoxycoformycin, diethylstilbestrol (DES), doxorubicin, etoposide(VP-16), epirubicin, esorubicin, fluorouracil (5-FU, Adrucil),gemcitabine, hexamethylmelamine, hydroxyprogesterone, hydroxyurea,idarubicin, ifosfamide, irinotecan, mafosfamide, melphalan, methotrexate(MTX), methylcyclohexylnitrosurea, mithramycin, mitomycin C,mitoxantrone, nitrogen mustards, paclitaxel (Taxol®),pentamethylmelamine, prednisone, procarbazine, tamoxifen, taxol,teniposide, testosterone, trimetrexate, topotecan, vincristine, andvinblastine.

In one embodiment, the subject is HPV+, and the subject has analteration (e.g., a mutation) in a gene in the PI3K(Phosphatidylinositol-3 kinase) pathway, such as mutation in a PIK3CA(phosphoinositide-3-kinase, catalytic, alpha polypeptide) gene, a PTEN(phosphatase and tensin homolog) gene, or an STK11 (serine/threoninekinase 11) gene. In one embodiment, said HPV+ patient can be treatedwith an inhibitor of a protein in the PI3K pathway, such as a PIK3CAprotein, a PTEN protein, or an STK11 protein, or a protein whoseexpression is altered (e.g., upregulated) as a result of a mutation in agene of the PI3K pathway. For example, the patient can be treated withan mTOR (mammalian Target of Rapamycin) inhibitor, such as rapamycin(sirolimus) or a rapamycin derivative (40-O-(2-hydroxyethyl) rapamycin,40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin or40-epi-(tetrazolyl)-rapamycin); a PI3K inhibitor, such as BKM120(Novartis, Basel, Switzerland), LY294002, or wortmannin; or a PI3K-mTORinhibitor, such as BEZ235 (Novartis, Basel, Switzerland), BGT226(Novartis, Basel, Switzerland) or XL765 (Exelixis, San Francisco,Calif.). In one embodiment, the subject is tested by genomic profilingto confirm upregulation of a PIK3CA gene or PTEN gene or STK11 geneprior to treatment with an mTOR inhibitor or PI3K inhibitor.

In one embodiment, the HPV− subject is determined to have an alterationin a gene in the PI3K pathway, such as mutation in a PIK3CA gene, a PTEN(phosphatase and tensin homolog) gene, or an STK11 (serine/threoninekinase 11) gene, and the subject is treated with an mTOR inhibitor, aPI3K inhibitor, or a PI3K-mTOR inhibitor. The mutation in the gene inthe PI3K pathway can be determined, for example, by genomic profiling.

In one aspect, the invention features a method of treating a subjecthaving a cancer, e.g., a head and neck cancer (e.g., an HNSCC). Themethod includes:

acquiring knowledge of a presence of HPV in the subject, and

responsive to a determination of the presence or absence of HPV in thesubject, selecting one or more of:

(1) identifying or selecting the subject as likely or unlikely torespond to a treatment;(2) selecting a treatment option, such as treatment with a CDKinhibitor, such as a CDK4/6 inhibitor; and/or(3) treating the subject, e.g., with the CDK inhibitor.

In one embodiment, the subject is determined to be HPV−, and responsiveto the determination that the patient is HPV−, the patient is identifiedas likely to respond to treatment with a CDK inhibitor. In anotherembodiment, the subject is determined to be HPV−, and responsive to thedetermination that the patient is HPV−, a cell-cycle inhibitor isselected as a treatment option. The cell-cycle inhibitor can be, forexample, a cyclin-dependent kinase (CDK) inhibitor, such as a CDK4 orCDK6 inhibitor, e.g., a CDK4/6 inhibitor, such as LEE011 (Novartis),LY-2835219 or PD 0332991 (Pfizer). In one embodiment, the CDK inhibitorinhibits CDK1, CDK2, CDK7, and/or CDK9. In certain embodiments, the CDKinhibitor is flavopiridol, indisulam, AZD5438, SNS-032, SCH 727965(Dinaciclib), JNJ-7706621, indirubin, or seliciclib. A CDK4/6 inhibitorcan inhibit both CDK4 and CDK6 activity. In one embodiment, the CDKinhibitor is not flavopiridol.

In another embodiment, the subject is further administered the selectedtreatment option.

In one embodiment, the subject is determined to be HPV+, and responsiveto the determination that the patient is HPV+, the patient is identifiedas unlikely to respond to treatment with a cell-cycle inhibitor. In oneembodiment, the subject is determined to be HPV+, and responsive to thedetermination that the patient is HPV+, an anti-cancer agent other thana CDK inhibitor is selected as a treatment option. For example, theanti-cancer agent is one or more of 4-hydroxyperoxycyclophosphoramide,5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), 5-azacytidine,6-mercaptopurine, 6-thioguanine, actinomycin D, amsacrine,bis-chloroethylnitrosurea, bleomycin, bryostatin-1, busulfan,carboplatin (Paraplatin®), chlorambucil, cisplatin (Platinol®),cetuximab (Erbitux®), colchicine, cyclophosphamide, cytarabine, cytosinearabinoside, dacarbazine, daunorubicin, daunomycin, dactinomycin,deoxycoformycin, diethylstilbestrol (DES), doxorubicin, etoposide(VP-16), epirubicin, esorubicin, fluorouracil (5-FU, Adrucil),gemcitabine, hexamethylmelamine, hydroxyprogesterone, hydroxyurea,idarubicin, ifosfamide, irinotecan, mafosfamide, melphalan, methotrexate(MTX), methylcyclohexylnitrosurea, mithramycin, mitomycin C,mitoxantrone, nitrogen mustards, paclitaxel (Taxol®),pentamethylmelamine, prednisone, procarbazine, tamoxifen, taxol,teniposide, testosterone, trimetrexate, topotecan, vincristine, andvinblastine.

In one embodiment, the subject, e.g., an HPV+ patient, is administeredradiation therapy, or is administered a surgery to treat the cancer. Insome embodiments, the patient is administered one or more of a radiationtherapy, a surgery to treat a cancer or an anti-cancer agent that is nota CDK inhibitor.

In one embodiment, the HPV− patient is further administered radiationtherapy, or is further administered a surgery to treat the cancer.

In certain embodiments, the subject has a localized cancer, e.g., alocalized cancer of the head or neck. In other embodiments, the subjecthas metastatic cancer. In certain embodiments, the subject is furtherevaluated for the presence of one or more of the alterations, e.g.,mutations, disclosed herein. In one embodiment, the treatment of thesubject is modified, e.g., decreased, discontinued, or otherwisealtered, in response to the detection of one or more of the alterations,e.g., mutations, described herein.

In one embodiment, the HPV− patient is further determined to have analteration, e.g., a mutation, in a cell cycle gene, such as a CDKN2A,CDKN2B or CCND1 gene. In one embodiment, the HPV− patient has aloss-of-function mutation in one or both of the CDKN2A gene or theCDKN2B gene, and in another embodiment, the HPV− patient has again-of-function mutation in the CCND1 gene.

In one embodiment, the subject, e.g., the HPV+ patient, is furtherdetermined to have a mutation in the PI3K pathway. For example, the HPV+patient can be determined to have a mutation in a PIK3CA gene, a PTENgene, or an STK11 gene. The determination can be made, for example, bygenomic profiling. In one embodiment, an HPV+ patient determined to havea mutation in a PIK3CA gene, a PTEN gene, or an STK11 gene can betreated with an inhibitor of a PIK3CA gene, a PTEN gene, or an STK11gene. For example, the patient can be treated with an mTOR inhibitor, aPI3K inhibitor, or a dual PI3K-mTOR inhibitor

In one embodiment, an HPV− patient, is determined to have a mutation inthe PI3K pathway, e.g., a mutation in a PIK3CA gene, a PTEN gene, or anSTK11 gene, and the HPV− patient is treated with an inhibitor of aPIK3CA gene, a PTEN gene, or an STK11 gene. The determination is made,for example, by genomic profiling.

In one aspect, the invention features a method of treating a subjecthaving a cancer, such as a head and neck cancer, e.g., an HNSCC. Themethod includes, for example, selecting a subject having a squamous cellcarcinoma of the head and neck on the basis of whether the subject isHPV− or HPV+, and if the subject is HPV−, then the subject isadministered a CDK inhibitor, and if the subject is HPV⁺, then thesubject is administered an anti-cancer agent other than a CDK inhibitor.

In one embodiment, the subject is HPV− and the CDK inhibitor is a CDK4or CDK6 inhibitor, e.g., a CDK4/6 inhibitor, such as LEE011 (Novartis),LY2835219, or PD 0332991 (Pfizer). In another embodiment, the CDKinhibitor inhibits CDK1, CDK2, CDK7, and/or CDK9. In certainembodiments, the CDK inhibitor is flavopiridol, indisulam, AZD5438,SNS-032, SCH 727965 (Dinaciclib), JNJ-7706621, indirubin or seliciclib.In one embodiment, the CDK inhibitor is not flavopiridol.

In another embodiment, the subject is HPV+, and one or more of thefollowing anti-cancer agents is administered to the subject:4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), 5-azacytidine, 6-mercaptopurine,6-thioguanine, actinomycin D, amsacrine, bis-chloroethylnitrosurea,bleomycin, bryostatin-1, busulfan, carboplatin (Paraplatin®),chlorambucil, cisplatin (Platinol®), cetuximab (Erbitux®), colchicine,cyclophosphamide, cytarabine, cytosine arabinoside, dacarbazine,daunorubicin, daunomycin, dactinomycin, deoxycoformycin,diethylstilbestrol (DES), doxorubicin, etoposide (VP-16), epirubicin,esorubicin, fluorouracil (5-FU, Adrucil), gemcitabine,hexamethylmelamine, hydroxyprogesterone, hydroxyurea, idarubicin,ifosfamide, irinotecan, mafosfamide, melphalan, methotrexate (MTX),methylcyclohexylnitrosurea, mithramycin, mitomycin C, mitoxantrone,nitrogen mustards, paclitaxel (Taxol®), pentamethylmelamine, prednisone,procarbazine, tamoxifen, taxol, teniposide, testosterone, trimetrexate,topotecan, vincristine, or vinblastine.

In one embodiment, the HPV+ patient is administered radiation therapy,or is administered a surgery to treat the cancer. In some embodiments,the patient is administered one or more of a radiation therapy, asurgery to treat a cancer or an anti-cancer agent that is not a CDKinhibitor.

In one embodiment, the HPV− patient is further administered radiationtherapy, or is further administered a surgery to treat the cancer.

In certain embodiments, the subject has a localized cancer, e.g., alocalized cancer of the head or neck. In other embodiments, the subjecthas metastatic cancer. In certain embodiments, the subject is furtherevaluated for the presence of one or more of the alterations, e.g.,mutations, disclosed herein. In one embodiment, the treatment of thesubject is modified, e.g., decreased, discontinued, or otherwisealtered, in response to the detection of one or more of the alterations,e.g., mutations, described herein.

In one embodiment, the HPV− patient is further determined to have agenomic alteration in a cell cycle gene, such as a CDKN2A gene, CDKN2Bgene, CCNE1 gene, CCND1 gene, CCND2 gene, CCND3 gene, CDK4 gene or CDK6gene. The genomic alteration can be, for example, a base substitution, asmall insertion, a gene amplification, or a gene deletion, such as adeletion of an entire gene, or both alleles of a gene, as in ahomozygous deletion. In one embodiment, the alteration in the CDKN2A orCDKN2B gene is a loss-of-function mutation, and in another embodiment,the alteration in the CCND1 gene is a gain-of-function mutation. Incertain embodiments, the alteration is an alteration listed in Table 4.

In one embodiment, the subject, e.g., HPV+ patient, is furtherdetermined to have a genomic alteration (e.g., a mutation) in the PI3Kpathway. For example, the HPV+ patient can be determined to have amutation in a PIK3CA gene, a PTEN gene, or an STK11 gene. In oneembodiment, said HPV+ patient can be treated with an inhibitor of aPIK3CA gene, a PTEN gene, or an STK11 gene. For example, the patient canbe treated with an mTOR inhibitor, a PI3K inhibitor, or a PI3K-mTORinhibitor.

In one embodiment, an HPV− subject is determined to have an alterationin a gene in the PI3K pathway, such as a mutation in a PIK3CA gene, aPTEN (phosphatase and tensin homolog) gene, or an STK11(serine/threonine kinase 11) gene, and the subject is furtheradministered an mTOR inhibitor, a PI3K inhibitor, or a PI3K-mTORinhibitor.

In one aspect, the invention features a method of treating subjecthaving a cancer, such as head and neck cancer, e.g., an HNSCC. Themethod includes, for example, selecting a subject having a cancer on thebasis of whether the subject is HPV+ or HPV⁻; acquiring knowledge ofwhether a subject has a mutation in a cell-cycle gene; and if thesubject has a mutation in a cell cycle gene, administering a CDKinhibitor to the subject. If the subject does not have a mutation in acell-cycle gene, then an anti-cancer drug other than a CDK inhibitor isadministered to the subject.

In one embodiment, the CDK inhibitor inhibits one or both of a CDK4 orCDK6 inhibitor, e.g., an inhibitor that inhibits CDK4 and CDK6, e.g., aCDK4/6 inhibitor. Exemplary CDK4/6 inhibitors include, e.g., LEE011(Novartis), LY-2835219, and PD 0332991 (Pfizer). In one embodiment, theCDK inhibitor inhibits CDK1, CDK2, CDK7, and/or CDK9. In certainembodiments, the CDK inhibitor is flavopiridol, indisulam, AZD5438,SNS-032, SCH 727965 (Dinaciclib), JNJ-7706621, indirubin, or seliciclib.In one embodiment, the CDK inhibitor is not flavopiridol.

In one embodiment, the anti-cancer agent other than a cell-cycleinhibitor is 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), 5-azacytidine, 6-mercaptopurine,6-thioguanine, actinomycin D, amsacrine, bis-chloroethylnitrosurea,bleomycin, bryostatin-1, busulfan, carboplatin (Paraplatin®),chlorambucil, cisplatin (Platinol®), cetuximab (Erbitux®), colchicine,cyclophosphamide, cytarabine, cytosine arabinoside, dacarbazine,daunorubicin, daunomycin, dactinomycin, deoxycoformycin,diethylstilbestrol (DES), doxorubicin, etoposide (VP-16), epirubicin,esorubicin, fluorouracil (5-FU, Adrucil), gemcitabine,hexamethylmelamine, hydroxyprogesterone, hydroxyurea, idarubicin,ifosfamide, irinotecan, mafosfamide, melphalan, methotrexate (MTX),methylcyclohexylnitrosurea, mithramycin, mitomycin C, mitoxantrone,nitrogen mustards, paclitaxel (Taxol®), pentamethylmelamine, prednisone,procarbazine, tamoxifen, taxol, teniposide, testosterone, trimetrexate,topotecan, vincristine, or vinblastine.

In one embodiment, the HPV+ patient is administered radiation therapy,or is administered a surgery to treat the cancer. In some embodiments,the patient is administered one or more of a radiation therapy, asurgery to treat a cancer or an anti-cancer agent that is not a CDKinhibitor.

In one embodiment, the HPV− patient is further administered radiationtherapy, or is further administered a surgery to treat the cancer.

In certain embodiments, the subject has a localized cancer, e.g., alocalized cancer of the head or neck. In other embodiments, the subjecthas metastatic cancer. In certain embodiments, the subject is furtherevaluated for the presence of one or more of the alterations, e.g.,mutations, disclosed herein. In one embodiment, the treatment of thesubject is modified, e.g., decreased, discontinued, or otherwisealtered, in response to the detection of one or more of the alterations,e.g., mutations, described herein.

In one embodiment, the HPV− patient is further determined to have amutation in a cell cycle gene, such as a CDKN2A or CDKN2B gene or aCCND1 gene. In one embodiment, the HPV− patient has a loss-of-functionmutation in a CDKN2A gene or a CDKN2B gene, and in another embodiment,the HPV− patient has a gain-of-function mutation in a CCND1 gene. In oneembodiment, the HPV− patient has a mutation listed in Table 4.

In one embodiment, the HPV+ patient is further determined to have amutation in the PI3K pathway. For example, the HPV+ patient can bedetermined to have a mutation in a PIK3CA gene, a PTEN gene, or an STK11gene. In one embodiment, the patient is treated with an mTOR inhibitor,a PI3K inhibitor, or a PI3K-mTOR inhibitor.

In one embodiment, an HPV− subject is determined to have an alterationin a gene in the PI3K pathway, such as a mutation in a PIK3CA gene, aPTEN (phosphatase and tensin homolog) gene, or an STK11(serine/threonine kinase 11) gene, and the subject is furtheradministered an mTOR inhibitor, a PI3K inhibitor, or a PI3K-mTORinhibitor. In another aspect, the invention features a method ofevaluating a subject (e.g., a patient) who has a cancer, such as a headand neck cancer, e.g., an HNSCC, e.g., for an appropriate therapy totreat the cancer. The method includes: acquiring information orknowledge of the presence of HPV in the subject (e.g., acquiringinformation from a sample from the subject that identifies a HPV asbeing present in the subject); wherein:

the presence of HPV indicates that the patient is less likely to respondto treatment with an agent that targets a cell cycle gene, e.g., a CDKinhibitor; and/or

the absence of HPV indicates that the patient is more likely to respondto treatment with an agent that targets a cell cycle gene, e.g., a CDKinhibitor.

The method can further include the step(s) of identifying (e.g.,evaluating, diagnosing, screening, and/or selecting) the subject asbeing positively correlated with increased risk for, or having, a cancerassociated with the mutant cell cycle gene. In one embodiment, thesubject is identified or selected as likely or unlikely to respond to atreatment, e.g., treatment with a cell-cycle inhibitor.

The method can further include treating the subject with an anti-canceragent, e.g., an anti-cancer agent as described herein.

In one embodiment, the CDK inhibitor inhibits one or both of a CDK4 orCDK6 inhibitor, e.g., inhibits CDK4 and CDK6, e.g., a CDK4/6 inhibitor.Exemplary CDK4/6 inhibitors include, e.g., LEE011 (Novartis), LY-2835219and PD 0332991 (Pfizer). In one embodiment, the CDK inhibitor inhibitsCDK1, CDK2, CDK7, and/or CDK9. In certain embodiments, the CDK inhibitoris flavopiridol, indisulam, AZD5438, SNS-032, SCH 727965 (Dinaciclib),JNJ-7706621, indirubin, or seliciclib. In one embodiment, the CDKinhibitor is not flavopiridol.

In one embodiment, the anti-cancer agent other than a cell-cycleinhibitor is 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), 5-azacytidine, 6-mercaptopurine,6-thioguanine, actinomycin D, amsacrine, bis-chloroethylnitrosurea,bleomycin, bryostatin-1, busulfan, carboplatin (Paraplatin®),chlorambucil, cisplatin (Platinol®), cetuximab (Erbitux®), colchicine,cyclophosphamide, cytarabine, cytosine arabinoside, dacarbazine,daunorubicin, daunomycin, dactinomycin, deoxycoformycin,diethylstilbestrol (DES), doxorubicin, etoposide (VP-16), epirubicin,esorubicin, fluorouracil (5-FU, Adrucil), gemcitabine,hexamethylmelamine, hydroxyprogesterone, hydroxyurea, idarubicin,ifosfamide, irinotecan, mafosfamide, melphalan, methotrexate (MTX),methylcyclohexylnitrosurea, mithramycin, mitomycin C, mitoxantrone,nitrogen mustards, paclitaxel (Taxol®), pentamethylmelamine, prednisone,procarbazine, tamoxifen, taxol, teniposide, testosterone, trimetrexate,topotecan, vincristine, or vinblastine.

The method can further include acquiring, e.g., directly or indirectly,a sample from a patient and evaluating the sample for the presence of amutant gene as described herein.

In one embodiment, a subject having a cancer, e.g., a head and necksquamous cell carcinoma (HNSCC), is evaluated for the responsiveness toan agent that targets a cell-cycle gene. For example, a subjectidentified as HPV− who is administered a CDK inhibitor can be monitoredat regular intervals, e.g., monthly, or once every three, six, 8 or 12months, or at shorter or longer intervals, for a response to the cancertreatment. If during the course of therapy, the subject fails to respondto therapy, the patient can be administered an alternative, course oftherapy.

In one embodiment, a subject having a cancer, e.g., a head and necksquamous cell carcinoma (HNSCC), who is positive or negative of HPV, isevaluated for the presence of a mutant gene, such as a mutant cell cyclegene. A subject identified as HPV− and as not having a mutant cell cyclegene can be administered a chemotherapeutic agent other than an agentthat targets a mutant cell cycle gene, or a gene or gene productdownstream of the cell cycle gene, and the patient can be monitored atregular intervals, e.g., monthly, or once every three, six, 8 or 12months, or at shorter or longer intervals, for the presence of a mutantcell cycle gene. If during the course of therapy, the subject is foundto carry a mutation in a cell cycle gene, the subject can stop receivingthe first therapy, or can be administered a decreased dose of the firsttherapy.

In certain embodiments, the subject is a patient or patient populationthat has participated in a clinical trial. In one embodiment, thesubject has participated in a clinical trial for evaluating a CDKinhibitor. In one embodiment, the clinical trial is discontinued orterminated. In one embodiment, the subject responded favorably to theclinical trial, e.g., experienced an improvement in at least one symptomof a cancer (e.g., decreased in tumor size, rate of tumor growth,increased survival). In other embodiments, the subject did not respondin a detectable way to the clinical trial. In one embodiment, thepresence or absence of HPV identifies the patient as being a candidateto receive treatment with a CDK inhibitor, or an anti-cancer agent thatis not a CDK inhibitor. For example, if a patient is HPV−, the patientis identified as a candidate to receive treatment with a CDK inhibitor.In another embodiment, the further identification of the presence of amutant cell cycle gene, such as a mutant CDKN2A gene or CDKN2B gene, ora mutant CCND1 gene, further confirms that the patient is a candidate toreceive treatment with a CDK inhibitor.

In a related aspect, a method of evaluating a patient or a patientpopulation is provided. The method includes: identifying, selecting, orobtaining information or knowledge that the patient or patientpopulation has participated in a clinical trial; acquiring informationor knowledge of the presence of HPV in the patient or patientpopulation; optionally acquiring genotype information of the subjectthat identifies a mutant gene, e.g., a cell cycle gene or PI3K pathwaygene as being present in the subject; optionally acquiring a sequencefor a nucleic acid molecule identified herein (e.g., a nucleic acidmolecule that includes a mutant sequence in a cell cycle gene); ordetecting the presence of a mutant nucleic acid or polypeptide in thesubject, wherein the absence of HPV in the patient identifies thepatient or patient population as a candidate to receive treatment with aCDK inhibitor, and wherein the presence of HPV identifies the patient orpatient population as a candidate to receive treatment with ananti-cancer agent other than a CDK inhibitor.

In certain embodiments, the subject is a patient or patient populationthat has participated in a clinical trial. In one embodiment, thesubject has participated in a clinical trial for evaluating a cell-cycleinhibitor. In one embodiment, the clinical trial is discontinued orterminated. In one embodiment, the subject responded favorably to theclinical trial, e.g., experienced an improvement in at least one symptomof a cancer (e.g., decreased in tumor size, rate of tumor growth,increased survival). In other embodiments, the subject did not respondin a detectable way to the clinical trial.

In some embodiments, the method further includes identifying the patientas HPV− and treating the subject with a cell cycle inhibitor, e.g., aninhibitor of CDK4 or CDK6, such as described herein, or identifying thepatient as HPV+ and treating the subject with an anti-cancer agent otherthan a cell cycle inhibitor, such as cisplatin (Platinol), Cetuximab(Erbitux), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin), orpaclitaxel (Taxol).

In yet another aspect, the invention features a method of evaluating apatient. The method includes:

selecting a patient on the basis that the patient has participated in aclinical trial or has been treated for a cancer, such as a head and neckcancer (e.g., HNSCC);

acquiring information that identifies the patient as HPV+ or HPV−, where

(i) identification of the subject as being HPV− identifies the patientas more likely to have improved cancer symptoms following treatment witha CDK inhibitor, and/or

(ii) identification of the subject as being HPV+ identifies the patientas being less likely to have improved cancer symptoms followingtreatment with a CDK inhibitor.

In one embodiment, the subject is determined to be HPV−, and the subjectis further administered a CDK inhibitor. In another embodiment, thesubject is determined to be HPV+, and the subject is furtheradministered an anti-cancer agent other than a CDK inhibitor.

In one embodiment, the CDK inhibitor inhibits one or both of a CDK4 orCDK6 inhibitor, e.g., inhibits CDK4 and CDK6, e.g., a CDK4/6 inhibitor.Exemplary CDK4/6 inhibitors include, e.g., LEE011 (Novartis), LY-2835219and PD 0332991 (Pfizer). In one embodiment, the CDK inhibitor inhibitsCDK1, CDK2, CDK7, and/or CDK9. In certain embodiments, the CDK inhibitoris flavopiridol, indisulam, AZD5438, SNS-032, SCH 727965 (Dinaciclib),JNJ-7706621, indirubin, or seliciclib.

In one embodiment, the anti-cancer agent other than a cell-cycleinhibitor is 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), 5-azacytidine, 6-mercaptopurine,6-thioguanine, actinomycin D, amsacrine, bis-chloroethylnitrosurea,bleomycin, bryostatin-1, busulfan, carboplatin (Paraplatin®),chlorambucil, cisplatin (Platinol®), cetuximab (Erbitux®), colchicine,cyclophosphamide, cytarabine, cytosine arabinoside, dacarbazine,daunorubicin, daunomycin, dactinomycin, deoxycoformycin,diethylstilbestrol (DES), doxorubicin, etoposide (VP-16), epirubicin,esorubicin, fluorouracil (5-FU, Adrucil), gemcitabine,hexamethylmelamine, hydroxyprogesterone, hydroxyurea, idarubicin,ifosfamide, irinotecan, mafosfamide, melphalan, methotrexate (MTX),methylcyclohexylnitrosurea, mithramycin, mitomycin C, mitoxantrone,nitrogen mustards, paclitaxel (Taxol®), pentamethylmelamine, prednisone,procarbazine, tamoxifen, taxol, teniposide, testosterone, trimetrexate,topotecan, vincristine, or vinblastine.

In one embodiment, the HPV+ patient is administered radiation therapy,or is administered a surgery to treat the cancer. In some embodiments,the patient is administered one or more of a radiation therapy, asurgery to treat a cancer or an anti-cancer agent that is not a CDKinhibitor.

In one embodiment, the HPV− patient is further administered radiationtherapy, or is further administered a surgery to treat the cancer.

In one embodiment, the HPV− patient is further determined to have amutation in a cell cycle gene, such as a CDKN2A or CDKN2B gene, or aCCND1 gene. In one embodiment, the HPV− patient has a mutation in theCDKN2A gene or CDKN2B gene, and in another embodiment, the HPV− patienthas a gain-of-function mutation in the CCND1 gene. In one embodiment,the HPV− patient is determined to have a mutation listed in Table 4.

In one embodiment, the HPV+ patient is further determined to have amutation in the PI3K pathway. For example, the HPV+ patient can bedetermined to have a mutation in a PIK3CA gene, a PTEN gene, or an STK11gene. In one embodiment, the patient is treated with an mTOR inhibitor,a PI3K inhibitor, or a PI3K-mTOR inhibitor.

In one embodiment, an HPV− subject is determined to have an alterationin a gene in the PI3K pathway, such as a mutation in a PIK3CA gene, aPTEN (phosphatase and tensin homolog) gene, or an STK11(serine/threonine kinase 11) gene, and the subject is furtheradministered an mTOR inhibitor, a PI3K inhibitor, or a PI3K-mTORinhibitor.

Methods described herein can include providing a report, such as inelectronic, web-based, or paper form, to the cancer patient, e.g. to theHNSCC patient, or to another person or entity, e.g., a caregiver, e.g.,a physician, e.g., an oncologist, a hospital, clinic, third-party payor,insurance company or government office. The report can include outputfrom the method, e.g., the indication of presence or absence of a HPV ina patient with HNSCC as described herein. In one embodiment, a report isgenerated, such as in paper or electronic form, which identifies thepresence or absence of HPV in a patient, and optionally, a recommendedcourse of therapy. In one embodiment, the report includes an identifierfor the patient from which the sequence was obtained. In one embodiment,the report is in web-based form.

The report can also include information on the HPV status and/or therole of a sequence, e.g., a mutant gene, such as a mutant cell cyclegene, as described herein. Such information can include information onprognosis, resistance, or potential or suggested therapeutic options.The report can include information on the likely effectiveness of atherapeutic option, the acceptability of a therapeutic option, or theadvisability of applying the therapeutic option to a patient, e.g., apatient having a sequence, alteration or mutation identified in thetest, and in embodiments, identified in the report. For example, thereport can include information, or a recommendation on, theadministration of a drug, e.g., the administration of a preselecteddosage or in a preselected treatment regimen, e.g., in combination withother drugs, to the patient. In an embodiment, not all mutationsidentified in the method are identified in the report. For example, thereport can be limited to mutations in genes having a preselected levelof correlation with the occurrence, prognosis, stage, or susceptibilityof the cancer to treatment, e.g., with a preselected therapeutic option.The report can be delivered, e.g., to an entity described herein, within7, 14, or 21 days from receipt of the sample by the entity practicingthe method.

Thus, in yet another aspect, the invention features a method forgenerating a personalized cancer treatment report. The method includes:

acquiring, e.g., obtaining, a sample from a subject having a cancer,e.g., a head and neck cancer (e.g., an HNSCC), for example, bydetermining whether the subject is HPV− or HPV+, and

selecting a treatment based on the whether the subject is HPV− or HPV+.In one embodiment, the subject is HPV−, and a CDK inhibitor is selectedas a treatment. In another embodiment, the subject is HPV+, and ananti-cancer agent other than a CDK inhibitor is selected as a treatment.In another embodiment, the subject, e.g., a patient, is furtheradministered the selected method of treatment.

In one embodiment, the method further includes providing a report toanother party. The other party can be, for example, the subject, acaregiver, a physician, an oncologist, a hospital, clinic, third-partypayor, insurance company or a government office. In some embodiments,the report is in electronic, web-based, or paper form. In oneembodiment, the report, or a separate report, identifies the presence orabsence of HPV in the subject, and optionally includes an identifier forthe subject from which the information was obtained. For example, thereport can contain one or more of the following: (i) information on theHPV status of the subject; (ii) information on prognosis, resistance, orpotential or suggested therapeutic options; (iii) information on thelikely effectiveness of a therapeutic option, (iv) the acceptability ofa therapeutic option, or the advisability of applying the therapeuticoption to a patient; or (v) information, or a recommendation on, theadministration of a drug.

In one embodiment, the subject is determined to be HPV− and a CDKinhibitor is selected for treating the subject. In another embodiment,the subject is determined to be HPV+ and an anti-cancer agent other thana CDK inhibitor is selected for treating the subject.

Alternatively, or in combination with, the aforesaid method ofgenerating a report, the method includes:

acquiring (e.g., obtaining) a sample, e.g., a tumor sample, from asubject having HNSCC, for example, detecting the presence of analteration as described herein in the sample, and

selecting a treatment based on the alteration (e.g., the mutation)identified. In one embodiment, the report is generated that annotatesthe selected treatment, or that lists, e.g., in order of preference, twoor more treatment options based on the mutation identified. In anotherembodiment, the subject, e.g., a patient, is further administered theselected method of treatment.

In one embodiment, a report is generated to memorialize each time apatient is tested for the presence of HPV, or for the presence of amutation in a cell cycle gene. For example, a patient who is determinednot to have HPV can be administered a cell cycle inhibitor, such as aCDK inhibitor to treat an HNSCC. The patient can be reevaluated atintervals, such as every month, every two months, every six months orevery year, or more or less frequently, to monitor the patient for animprovement in cancer symptoms. In some embodiments, if the patient doesnot respond to treatment with the CDK inhibitor, the patient's therapycan be adjusted to incorporate or substitute alternative cancertherapies. The report can record at least the treatment history of thepatient.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, and theexample are illustrative only and not intended to be limiting.

The details of one or more embodiments featured in the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages featured in the invention will beapparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 is a graph depicting the correlation between HPV status andclasses of gene mutations.

FIGS. 2A and 2B are a list of 182 genes sequenced across the entirecoding sequence (FIG. 2A), and 14 genes sequenced across selectedintrons (FIG. 2B).

FIGS. 3A and 3B are representations of hierarchical clustering of HPV+and HPV− HNSCC samples using all detected genetic changes (FIG. 3A) orexcluding mutations within TP53 (FIG. 3B).

FIG. 4 is a graph depicting that genetic changes in sample “P17 neg”detected by NGS.

FIG. 5 is Infinium CNV profiling of HPV+ and HPV− HNSCC samples.

FIG. 6 is a comparison of mutated pathways in HNSCC and lungadenocarcinoma.

FIGS. 7A and 7B are heat maps of genomic changes associated with patientcharacteristics and observed changes. Demographic and histologic dataare described above the heatmap.

DETAILED DESCRIPTION

The invention is based, at least in part, on the discovery that HPVstatus is predictive of the underlying genotype in subjects having acancer, such as a cancer of the head and neck, e.g., a head and necksquamous cell carcinoma (HNSCC). HNSCC patients who are HPV− are morelikely to carry mutations in cell cycle genes such as CDKN2A, CDKN2B,CCNE1, CCND1, CCND2, CCND3, CDK4 or CKD6, and are therefore more likelyto respond to treatment with a cell cycle inhibitor, such as a CDKinhibitor. Determination that the subject is HPV− is sufficient toconclude that the subject is a candidate to receive treatment with a CDKinhibitor, e.g., a CDK4/6 inhibitor. Further genomic profiling is notnecessary before making the determination that the subject is likely torespond to treatment with a CDK inhibitor, or before administering theCDK inhibitor.

Cancers known collectively as head and neck cancers usually begin in thesquamous cells that line the moist, mucosal surfaces inside the head andneck (for example, inside the mouth, the nose, and the throat). Thesesquamous cell cancers are often referred to as squamous cell carcinomasof the head and neck (or head and neck squamous cell carcinomas, HNSCC,which term is used herein). HNSCC includes squamous cell carcinomas ofthe mouth, nasopharynx (where the nasal cavity and the eustacian tubesconnect with the upper part of the throat), oropharynx (the middle partof the throat that includes the soft palate, the base of the tongue, andthe tonsils), hypopharynx (the pyriform sinuses, the posteriorpharyngeal wall and the postcricoid area), larynx, and trachea.

Squamous cell carcinomas of the mouth include, for example, cancers ofthe inner lip, tongue, floor of the mouth, gingivae, and hard palate.Squamous cell carcinomas of the head and neck can also begin in thesalivary glands.

There are more than 100 different types of human papillomaviruses (HPVs)and the genomes of over 80 different types have been completelysequenced. The current classification system for HPV is based onsimilarities in genomic sequence, and divides the HPVs into threeclinical categories: (i) anogenital or mucosal; (ii) nongenitalcutaneous; (iii) epidermodysplasia verruciformis (EV). The majority ofHPV-related HNSCCs are associated with type 16 HPV, with types 18, 31and 33 accounting for almost all of the remaining cases (Snow et al.,Adv. Anat. Pathol. 17:394-403, 2010).

The mucosal HPV infections are classified further as latent(asymptomatic), subclinical, or clinical. Clinical lesions are grosslyapparent, whereas latent infections are detected only with tests forviral DNA. Subclinical lesions are identified by application of 3-5%acetic acid and inspection under magnification. Most HPV infections arelatent; clinically apparent infections usually result in warts ratherthan malignancies.

Nongenital cutaneous infections include common warts, plantar warts,flat warts and other skin lesions.

Epidermodysplasia verruciformis (EV) is a rare, inherited disorder thatpredisposes patients to widespread human papillomavirus (HPV) infectionand cutaneous squamous cell carcinomas.

HPV can be detected by assays known in the art such as in situhybridization (ISH), immunohistochemistry (IHC), PCR, dot blot, reverseline blot, DNA enzyme immunoassay, Southern blot, sequencing, Northernblot, or Western blot analysis. The detection method can detect HPVprotein, DNA or RNA, e.g., mRNA, and specimen types evaluated can befrozen, fresh or FFPE (formalin fixed paraffin embedded) tissue; saliva;or cytological preparations.

In one embodiment, HPV is detected by IHC to detect the p16 protein(encoded by CDKN2A), a component of the retinoblastoma tumor suppressorpathway. The protein p16 is strongly and diffusely expressed in about93% of HPV-associated squamous cell carcinomas (SCCs) but is absent inHPV-negative carcinomas.

In other embodiments, PCR (polymerase chain reaction) or RFLP(restriction fragment length polymorphism) is performed to detect aspecific HPV type in a subject.

Accordingly, the invention provides, at least in part, methods oftreating a cancer, such as HNSCC, according to whether a subject is HPV−or HPV+. If the subject is HPV−, the subject is administered ananti-cancer agent that targets a cell cycle gene, such as a CDKinhibitor, to treat the cancer.

In one aspect, the invention features compositions and methods toidentify new CDK inhibitors; to treat or prevent a cancer, e.g., anHNSCC; as well as to methods and assays for evaluating a cancer (e.g.,evaluating one or more of: cancer progression, cancer treatment responseor resistance to cancer treatment; selection of a treatment option,stratification of a patient population, and/or more effectivemonitoring, treatment or prevention of cancer).

The cell cycle gene CDKN2A (Cyclin Dependent Kinase Inhibitor 2A)inhibits CDK4 in vivo. The invention is based, at least in part, on thediscovery that 50% to 60% of HNSCC patients who are HPV− also carry aloss-of-function mutation in the CDKN2A gene, which results in aberrantcell-cycle activity. Thus, HNSCC patients who are HPV− can beadministered a CDK inhibitor, such as a CDK4 or CDK6 inhibitor. TheHNSCC patient can optionally be tested for the presence of a mutation inthe CDKN2A gene. Without being bound by theory, loss-of-functionmutations in CDKN2A frequently resulted from copy number alterations,but a low frequency of point mutations was also observed.

The cell cycle gene CCND1 (Cyclin D1) encodes a protein that binds CDK4and inhibits CDK4/cyclin D1 enzymes in vivo. The invention is based, atleast in part, on the discovery that 60% to 70% of HNSCC patients whoare HPV− also carry a gain-of-function mutation in the CCND1 gene, whichresults in aberrant cell-cycle activity. Thus, HNSCC patients who areHPV− can be administered a CDK inhibitor, such as a CDK4 or CDK6inhibitor. The HNSCC patient can optionally be tested for the presenceof mutation in the CCND1 gene. The gain-of-function mutations in CCND1are typically the result of copy number alterations, but on rareoccasions, a point mutation may also cause a gain of function phenotype.

Other mutations found to correlate with HPV status are described inTables 1 and 4. The invention therefore also provides, at least in part,isolated nucleic acid molecules containing a mutation described in Table1 or Table 4, nucleic acid constructs, and host cells containing thenucleic acid molecules; purified mutant polypeptides comprising amutation described in Table 1 or Table 4, and binding agents, e.g.,antibodies and small molecule compounds that specifically bind themutant proteins. The invention also provides detection reagents (e.g.,probes, primers, antibodies, kits); screening assays for identifyingnovel inhibitors; as well as methods, assays and kits for evaluating,identifying, assessing and/or treating a subject having a cancer, e.g.,a cancer having a mutation disclosed herein.

Certain terms are defined. Additional terms are defined throughout thespecification.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity, e.g., a sample, or a value,e.g., a numerical value, or nucleic acid sequence or amino acidsequence, by “directly acquiring” or “indirectly acquiring” the physicalentity or value. “Directly acquiring” means performing a process (e.g.,performing a synthetic or analytical method, e.g., a sequencingreaction) to obtain the physical entity or value. “Indirectly acquiring”refers to receiving the physical entity or value from another party orsource (e.g., a third party laboratory that directly acquired thephysical entity or value). Directly acquiring a physical entity includesperforming a process that includes a physical change in a physicalsubstance, e.g., a starting material, or a nucleic acid sample.Exemplary changes include making a physical entity from two or morestarting materials, shearing or fragmenting a substance, separating orpurifying a substance, combining two or more separate entities into amixture, performing a chemical reaction that includes breaking orforming a covalent or non-covalent bond. Directly acquiring a valueincludes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

“Acquiring a sequence” as the term is used herein, refers to obtainingpossession of a nucleotide sequence or amino acid sequence, by “directlyacquiring” or “indirectly acquiring” the sequence. “Directly acquiring asequence” means performing a process (e.g., performing a synthetic oranalytical method) to obtain the sequence, such as performing asequencing method (e.g., a Next Generation Sequencing (NGS) method).“Indirectly acquiring a sequence” refers to receiving information orknowledge of, or receiving, the sequence from another party or source(e.g., a third party laboratory that directly acquired the sequence).The sequence acquired need not be a full sequence, e.g., sequencing ofat least one nucleotide, or obtaining information or knowledge, thatidentifies a mutation disclosed herein as being present in a subjectconstitutes acquiring a sequence.

Directly acquiring a sequence includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial, such as a tissue sample, e.g., a biopsy, or an isolatednucleic acid (e.g., DNA or RNA) sample. Exemplary changes include makinga physical entity from two or more starting materials, shearing orfragmenting a substance, such as a genomic DNA fragment; separating orpurifying a substance (e.g., isolating a nucleic acid sample from atissue); combining two or more separate entities into a mixture,performing a chemical reaction that includes breaking or forming acovalent or non-covalent bond. Directly acquiring a value includesperforming a process that includes a physical change in a sample oranother substance as described above.

“Acquiring a sample” as the term is used herein, refers to obtainingpossession of a sample, e.g., a tissue sample or nucleic acid sample, by“directly acquiring” or “indirectly acquiring” the sample. “Directlyacquiring a sample” means performing a process (e.g., performing aphysical method such as a surgery or extraction) to obtain the sample.“Indirectly acquiring a sample” refers to receiving the sample fromanother party or source (e.g., a third party laboratory that directlyacquired the sample). Directly acquiring a sample includes performing aprocess that includes a physical change in a physical substance, e.g., astarting material, such as a tissue, e.g., a tissue in a human patientor a tissue that has was previously isolated from a patient. Exemplarychanges include making a physical entity from a starting material,dissecting or scraping a tissue; separating or purifying a substance(e.g., a sample tissue or a nucleic acid sample); combining two or moreseparate entities into a mixture; performing a chemical reaction thatincludes breaking or forming a covalent or non-covalent bond. Directlyacquiring a sample includes performing a process that includes aphysical change in a sample or another substance, e.g., as describedabove.

“Binding entity” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of specifically bindingto an analyte. The binding entity can be an affinity tag on a nucleicacid sequence. In certain embodiments, the binding entity allows forseparation of the nucleic acid from a mixture, such as an avidinmolecule, or an antibody that binds to the hapten or an antigen-bindingfragment thereof. Exemplary binding entities include, but are notlimited to, a biotin molecule, a hapten, an antibody, an antibodybinding fragment, a peptide, and a protein.

“Complementary” refers to sequence complementarity between regions oftwo nucleic acid strands or between two regions of the same nucleic acidstrand. It is known that an adenine residue of a first nucleic acidregion is capable of forming specific hydrogen bonds (“base pairing”)with a residue of a second nucleic acid region which is antiparallel tothe first region if the residue is thymine or uracil. Similarly, it isknown that a cytosine residue of a first nucleic acid strand is capableof base pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.In certain embodiments, the first region comprises a first portion andthe second region comprises a second portion, whereby, when the firstand second portions are arranged in an antiparallel fashion, at leastabout 50%, at least about 75%, at least about 90%, or at least about 95%of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. In otherembodiments, all nucleotide residues of the first portion are capable ofbase pairing with nucleotide residues in the second portion.

The term “cancer” or “tumor” is used interchangeably herein. These termsrefer to the presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in theform of a tumor, but such cells can exist alone within an animal, or canbe a non-tumorigenic cancer cell, such as a leukemia cell. These termsinclude a solid tumor, a soft tissue tumor, or a metastatic lesion. Asused herein, the term “cancer” includes premalignant, as well asmalignant cancers.

Cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cancer isalso “inhibited” if recurrence or metastasis of the cancer is reduced,slowed, delayed, or prevented.

“Chemotherapeutic agent” means a chemical substance, such as a cytotoxicor cytostatic agent, that is used to treat a condition, particularlycancer.

As used herein, “cancer therapy” and “cancer treatment” are synonymousterms.

As used herein, “chemotherapy” and “chemotherapeutic” and“chemotherapeutic agent” are synonymous terms.

As used herein, a “cell-cycle gene” is a gene whose activity affectsregulation of the cell cycle, or whose expression levels varyperiodically with the cell-cycle.

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence similarity between two polynucleotide sequences orbetween two polypeptide sequences, with identity being a more strictcomparison. The phrases “percent identity or homology” and “% identityor homology” refer to the percentage of sequence similarity found in acomparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value there between.Identity or similarity can be determined by comparing a position in eachsequence that can be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical or matching nucleotides at positions sharedby the polynucleotide sequences. A degree of identity of polypeptidesequences is a function of the number of identical amino acids atpositions shared by the polypeptide sequences. A degree of homology orsimilarity of polypeptide sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences. The term“substantially identical,” as used herein, refers to an identity orhomology of at least 75%, at least 80%, at least 85%, at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

“Likely to” or “increased likelihood,” as used herein, refers to anincreased probability that an item, object, thing or person will occur.Thus, in one example, a subject that is likely to respond to treatmentwith a cell cycle inhibitor, such as a CDK inhibitor, has an increasedprobability of responding to treatment with the cell cycle inhibitor CDKinhibitor relative to a reference subject or group of subjects.

“Unlikely to” refers to a decreased probability that an event, item,object, thing or person will occur with respect to a reference. Thus, asubject that is unlikely to respond to treatment with a cell cycleinhibitor, such as a CDK inhibitor, has a decreased probability ofresponding to treatment with the cell cycle inhibitor relative to areference subject or group of subjects.

As used herein, “genomic profiling” means sequencing all or a part ofthe genome of a subject, such as to identify the nucleotide sequence ofgenes or a subset of genes in the subject, such as to identify genomicalterations (e.g., mutations) that would identify the subject as acandidate to receive certain drugs or other therapeutic agents. Genomicprofiling can be performed by a method described herein, such as by anext-generation sequencing method, or a massively parallel sequencingmethod.

“Sequencing” a nucleic acid molecule requires determining the identityof at least 1 nucleotide in the molecule. In embodiments, the identityof less than all of the nucleotides in a molecule are determined. Inother embodiments, the identity of a majority or all of the nucleotidesin the molecule is determined.

“Next-generation sequencing or NGS or NG sequencing” as used herein,refers to any sequencing method that determines the nucleotide sequenceof either individual nucleic acid molecules (e.g., in single moleculesequencing) or clonally expanded proxies for individual nucleic acidmolecules in a highly parallel fashion (e.g., greater than 10⁵ moleculesare sequenced simultaneously). In one embodiment, the relative abundanceof the nucleic acid species in the library can be estimated by countingthe relative number of occurrences of their cognate sequences in thedata generated by the sequencing experiment. Next generation sequencingmethods are known in the art, and are described, e.g., in Metzker, M.(2010) Nature Biotechnology Reviews 11:31-46, incorporated herein byreference. Next generation sequencing can detect a variant present inless than 5% of the nucleic acids in a sample.

“Sample,” “tissue sample,” “patient sample,” “patient cell or tissuesample” or “specimen” each refers to a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample can be solid tissue as from a fresh, frozen and/or preservedorgan, tissue sample, biopsy, or aspirate; blood or any bloodconstituents; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid or interstitial fluid; or cells from any time ingestation or development of the subject. The tissue sample can containcompounds that are not naturally intermixed with the tissue in naturesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics or the like. In one embodiment, the sample is preserved as afrozen sample or as formaldehyde- or paraformaldehyde-fixedparaffin-embedded (FFPE) tissue preparation. For example, the sample canbe embedded in a matrix, e.g., an FFPE block or a frozen sample.

A “tumor nucleic acid sample” as used herein, refers to nucleic acidmolecules from a tumor or cancer sample. Typically, it is DNA, e.g.,genomic DNA, or cDNA derived from RNA, from a tumor or cancer sample. Incertain embodiments, the tumor nucleic acid sample is purified orisolated (e.g., it is removed from its natural state).

A “control” or “reference” “nucleic acid sample” as used herein, refersto nucleic acid molecules from a control or reference sample. Typically,it is DNA, e.g., genomic DNA, or cDNA derived from RNA, not containingthe alteration or variation in the gene or gene product, e.g., notcontaining a mutation in a cell cycle gene. In certain embodiments, thereference or control nucleic acid sample is a wild-type or a non-mutatedsequence. In certain embodiments, the reference nucleic acid sample ispurified or isolated (e.g., it is removed from its natural state). Inother embodiments, the reference nucleic acid sample is from a non-tumorsample, e.g., a blood control, a normal adjacent tumor (NAT), or anyother non-cancerous sample from the same or a different subject.

Headings, e.g., (a), (b), (i) etc, are presented merely for ease ofreading the specification and claims. The use of headings in thespecification or claims does not require the steps or elements beperformed in alphabetical or numerical order or the order in which theyare presented.

Various aspects of the invention are described in further detail below.Additional definitions are set out throughout the specification.

Evaluation of Subjects

Subjects, e.g., patients, can be evaluated for the presence of an HPVmolecule. For example, a sample from a subject, such as a blood or urinesample, can be evaluated to identify an HPV nucleic acid or HPV proteinin the sample. For example, an HPV nucleic acid can be identified byPCR, Northern blot analysis, or sequencing of nucleic acids in thesample. HPV protein can be identified, e.g., by Western blot analysis. Asample from a subject can be, for example, a blood or serum sample, or aurine sample, or a tissue sample, such as a tumor tissue sample, such asfrom a biopsy, or a buccal swab.

In some embodiments, subjects, e.g., patients, can be evaluated for thepresence of a mutant gene. A patient can be evaluated, for example, bydetermining the genomic sequence of the patient, e.g., by an NGS method.Alternatively, or in addition, evaluation of a patient can includedirectly assaying for the presence of a mutant gene in the patient, suchas by an assay to detect a mutant nucleic acid (e.g., DNA or RNA), suchas by, Southern blot, Northern blot, or RT-PCR, e.g., qRT-PCR.Alternatively, or in addition, a patient can be evaluated for thepresence of a mutant protein, such as by immunohistochemistry, Westernblot, immunoprecipitation, or immunomagnetic bead assay.

Evaluation of a patient can also include a cytogenetic assay, such as byfluorescence in situ hybridization (FISH), to identify the chromosomalrearrangement resulting in the mutant gene. For example, to performFISH, at least a first probe tagged with a first detectable label can bedesigned to target a sequence in a functional domain of a gene.

Additional methods for mutant gene detection are provided below.

In one aspect, the results of a clinical trial, e.g., a successful orunsuccessful clinical trial, can be repurposed to identify agents thattarget a mutant gene. By one exemplary method, a candidate agent used ina clinical trial can be reevaluated to determine if the agent in thetrial targets a mutant gene, or is effective to treat a tumor containinga mutant gene. For example, subjects who participated in a clinicaltrial for an agent, e.g., a CDK inhibitor, can be identified. Patientswho experienced an improvement in symptoms, e.g., HSNCC symptoms, suchas decreased tumor size, or decreased rate of tumor growth, can beevaluated for the presence of a mutation in the ligand binding domain ofthe gene. Patients who did not experience an improvement in cancersymptoms can also be evaluated for the presence of a mutation, such asin a cell cycle gene. Where patients carrying a mutation in a functionaldomain of the gene are found to have been more likely to respond to thetest agent than patients who did not carry a mutation in the ligandbinding domain, then the agent is determined to be an appropriatetreatment option for a patient carrying the mutant gene.

“Reevaluation” of patients can include, for example, determining thegenomic sequence of the patients, or a subset of the clinical trialpatients, e.g., by an NGS method. Alternatively, or in addition,reevaluation of the patients can include directly assaying for thepresence of a gene mutation in the patient, such as by an assay todetect a mutant nucleic acid (e.g., RNA), such as by RT-PCR, e.g.,qRT-PCR. Alternatively, or in addition, a patient can be evaluated forthe presence of a mutant protein, such as by immunohistochemistry,Western blot, immunoprecipitation, or immunomagnetic bead assay.

Clinical trials suitable for repurposing as described above includetrials that tested CDK inhibitors, such as CDK4 or CKD6 inhibitors.

In one embodiment, the HPV molecule and/or mutant gene is detected priorto initiating, during, or after, a treatment in a subject. In oneembodiment, the HPV molecule and/or mutant gene is detected at the timeof diagnosis with a cancer. In other embodiments, the HPV moleculeand/or mutant gene is detected at a pre-determined interval, e.g., afirst point in time and at least at a subsequent point in time.

In certain embodiments, responsive to a determination of the presence ofthe HPV molecule and/or mutant gene, the method further includes one ormore of:

(1) stratifying a patient population (e.g., assigning a subject, e.g., apatient, to a group or class);

(2) identifying or selecting the subject as likely or unlikely torespond to a treatment, e.g., a treatment as described herein;

(3) selecting a treatment option, e.g., administering or notadministering a preselected therapeutic agent, e.g., an agent describedherein; or

(4) prognosticating the time course of the disease in the subject (e.g.,evaluating the likelihood of increased or decreased patient survival).

In certain embodiments, the therapeutic agent is a cell cycle inhibitor,such as flavopiridol; indisulam; AZD5438; SNS-032; PD 0332991; SCH727965 (Dinaciclib); LY-2835219; Seliciclib; LEE011 (Novartis);JNJ-7706621; indirubin; or PD 0332991 (Pfizer). In other embodiments,the cell-cycle inhibitor is a CDK inhibitor, such as flavopiridol;indisulam; AZD5438; SNS-032; PD 0332991; SCH 727965 (Dinaciclib);Seliciclib; JNJ-7706621; or indirubin. In yet other embodiments, thecell-cycle inhibitor is an inhibitor of CDK4 and/or CDK6, such as LEE011(Novartis); LY-2835219; or PD 0332991 (Pfizer).

In some embodiments, the therapeutic agent is other than a cell-cycleinhibitor, such as cisplatin (Platinol), Cetuximab (Erbitux),fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin), or paclitaxel(Taxol).

In certain embodiments, responsive to the determination of the presenceof the mutant gene, the subject is classified as a candidate to receivetreatment with an anti-cancer agent.

In one embodiment, responsive to the determination of the presence ofthe HPV molecule and/or the mutant gene, the subject, e.g., a patient,can further be assigned to a particular class if a mutant gene isidentified in a sample of the patient. In one embodiment, the subject,e.g., a patient, is assigned to a second class if the HPV moleculeand/or the mutation is not present.

In another embodiment, responsive to the determination of the presenceof the HPV molecule and/or mutant gene, the subject is identified aslikely to respond to a treatment as described herein.

In yet another embodiment, responsive to the determination of thepresence of the HPV molecule and/or mutant gene, the method includesadministering an anti-cancer agent as described herein to the subject.

In one embodiment, a subject who is determined not to carry the HPVmolecule and/or a mutant gene is reevaluated at intervals, such as everymonth, every two months, every six months or every year, or more or lessfrequently, to monitor the patient for the development of a mutation ingene, e.g., in a cell-cycle gene. For example, if a patient isdetermined not to carry a mutant gene, then the patient can bedetermined to be a candidate for treatment with a first agent, such asan anti-cancer agent that acts other than by inhibiting the cell cycle.If the patient is subsequently determined to have a mutant gene,administration of the first agent to the patient can be stopped, and thepatient can be administered a second agent, such as a cell-cycleinhibitor. In some embodiments, the patient continues to receivetreatment with the first agent, and optionally, the patient can receivemore frequent monitoring for a worsening of cancer symptoms.

Therapeutic Methods

In one embodiment, the invention features a method of treating apatient, such as an HPV− patient having a cancer, e.g., an HNSCC. Thepatient can have a tumor harboring a mutant gene as described herein,e.g., a mutant cell cycle gene such as a mutant CDKN2A or CDKN2B orCCND1 gene as described herein. The methods include administering ananti-cancer agent, e.g., a CDK inhibitor, alone or in combination, e.g.,in combination with other chemotherapeutic agents or procedures, in anamount sufficient to reduce or inhibit the tumor cell growth, and/ortreat or prevent the cancer(s), in the subject.

“Treat,” “treatment,” and other forms of this word refer to theadministration of a CDK inhibitor, alone or in combination with a secondagent to impede growth of a cancer, to cause a cancer to shrink byweight or volume, to extend the expected survival time of the subjectand or time to progression of the tumor or the like. In those subjects,treatment can include, but is not limited to, inhibiting tumor growth,reducing tumor mass, reducing size or number of metastatic lesions,inhibiting the development of new metastatic lesions, prolongedsurvival, prolonged progression-free survival, prolonged time toprogression, and/or enhanced quality of life.

As used herein, unless otherwise specified, the terms “prevent,”“preventing” and “prevention” contemplate an action that occurs before asubject begins to suffer from the re-growth of the cancer and/or whichinhibits or reduces the severity of the cancer.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of the cancer, or todelay or minimize one or more symptoms associated with the cancer. Atherapeutically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other therapeuticagents, which provides a therapeutic benefit in the treatment ormanagement of the cancer. The term “therapeutically effective amount”can encompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of the cancer, or enhances the therapeutic efficacyof another therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to preventre-growth of the cancer, or one or more symptoms associated with thecancer, or prevent its recurrence. A prophylactically effective amountof a compound means an amount of the compound, alone or in combinationwith other therapeutic agents, which provides a prophylactic benefit inthe prevention of the cancer. The term “prophylactically effectiveamount” can encompass an amount that improves overall prophylaxis orenhances the prophylactic efficacy of another prophylactic agent.

As used herein, the term “patient” or “subject” refers to an animal,typically a human (i.e., a male or female of any age group, e.g., apediatric patient (e.g, infant, child, adolescent) or adult patient(e.g., young adult, middle-aged adult or senior adult) or other mammal,such as a primate (e.g., cynomolgus monkey, rhesus monkey). When theterm is used in conjunction with administration of a compound or drug,then the patient has been the object of treatment, observation, and/oradministration of the compound or drug.

In certain embodiments, the cancer includes, but is not limited to, asolid tumor, a soft tissue tumor, and a metastatic lesion (e.g., acancer as described herein). In one embodiment, the cancer is a squamouscell carcinoma of the head and neck (HNSCC).

In other embodiments, the cancer is chosen from uterine or cervicalcancer or a tumor of the genitalia, such as the vulva, vagina or penis.In other embodiments the cancer is a testicular cancer, urinary bladdercancer, lung cancer, thyroid cancer, colorectal cancer, adenocarcinoma,melanoma, B cell cancer, bronchus cancer, cancer of the oral cavity orpharynx, cancer of hematological tissues, esophageal cancer,esophageal-gastric cancer, gastric cancer, kidney cancer, liver cancer,multiple myeloma, pancreatic cancer, salivary gland cancer, small bowelor appendix cancer, stomach cancer, inflammatory myofibroblastic tumors,gastrointestinal stromal tumor (GIST), and the like.

In certain embodiments, the cancer, e.g., HNSCC, is treated with a CDKinhibitor. In one embodiment, the CDK inhibitor inhibits one or both ofCDK4 or CDK6. In embodiments, the CDK inhibitor is an orally active,selective CDK4/6 inhibitor with ability to block retinoblastoma (Rb)phosphorylation. Exemplary CDK4/6 inhibitors are described in, e.g., WO2007/140222, WO 2010/020675, WO 2013/006368, WO 2013/006532, WO2011/130232, US 2013/0150342, W2011/101409, US 2013/184285,WO2006024945, WO2006024945, and EP1256578B1, all of which are herebyincorporated by reference in their entirety.

In one embodiment, the CDK inhibitor is chosen from flavopiridol,indisulam, AZD5438, SNS-032, SCH 727965 (Dinaciclib), Seliciclib,JNJ-7706621, or indirubin. In other embodiments, the CDK inhibitor isnot flavopiridol.

In another embodiment, the CDK4/6 inhibitor is chosen from LEE011(Novartis); LY-2835219 (Eli Lilly); or PD 0332991 (Pfizer).

In one embodiment, the CDK 4/6 inhibitor has the following structure:

also referred to herein as LEE011. In one embodiment, the CDK 4/6inhibitor has the following chemical name:4-(5-chloro-3-isopropyl-1H-pyrazol-4-yl)-N-(5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)pyrimidin-2-amine.

In another embodiment, the CDK 4/6 inhibitor has the followingstructure:

also referred to herein as LY-2835219. In one embodiment, the CDK 4/6inhibitor has the following chemical name:(N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine).

In yet another embodiment, the CDK 4/6 inhibitor has the followingstructure:

also referred to herein as PD 0332991. In one embodiment, the CDK 4/6inhibitor has the following chemical name:6-acetyl-8-cyclopentyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-onehydrochloride.

Further examples of publications describing the aforesaid inhibitors andtheir activities include Finn, R S et al. (2009) Breast Cancer Res.11(5):R77; Zhang, Y. in Proceedings of the AACR-NCI-EORTC InternationalConference: Molecular Targets and Cancer Therapeutics, 2011:10 (11Suppl): Abstract nr A236; Clinical Trial Gov. Identifier NCT01237236;and Clinical Trial Gov. Identifier NCT01394016, incorporated herein byreference.

In another embodiment, the CDK inhibitor is BAY1000394. BAY1000394 is anorally bioavailable CDK inhibitor. It inhibits the activity ofcell-cycle CDKs, including CDK1, CDK2, CDK3, CDK4, and oftranscriptional CDKs CDK7 and CDK9 with IC50 values in the range between5 and 25 nM. BAY1000394 has the chemical name: 2-Butanol,3-[[2-[[4-[[S(R)]-S-cyclopropylsulfonimidoyl]phenyl]amino]-5-(trifluoromethyl)-4-pyrimidinyl]oxy]-,(2R,3R)-; and has the following structure:

In another embodiment, the CDK inhibitor is ZK-304709. ZK-304709 is apotent multi-target tumor growth inhibitor. ZK-304709 inhibits theactivity of cell-cycle CDKs, including CDK1, CDK2, CDK4, and oftranscriptional CDKs CDK7 and CDK9, with IC50 values in the nanomolarrange. ZK-304709 also inhibits the activity of vascular endothelialgrowth factor receptor tyrosine kinases (VEGFRs), including VEGFR 1,VEGFR 2, and VEGFR3 and of platelet-derived growth factor receptor betatyrosine kinase (PDGFR). ZK-304709 has the chemical name:(Z)-3,3-dimethyl-2′-oxo-[2,3′-biindolinylidene]-5′-sulfonamide; and hasthe following structure:

In another embodiment, the CDK inhibitor is SNS032. SNS032 inhibits theactivity of cell-cycle CDKs, including CDK1, CDK2, and oftranscriptional CDKs CDK4, CDK7 and CDK9. SNS-032 has low sensitivity toCDK1 and CDK4 with IC50 of 480 nM and 925 nM, respectively. SNS032 hasthe chemical name:N-(5-((5-tert-butyloxazol-2-yl)methylthio)thiazol-2-yl)piperidine-4-carboxamide;and has the following structure:

In another embodiment, the CDK inhibitor is seliciclib. Seliciclibinhibits the activity of CDKs, including CDK2 and CDK5. Seliciclib hasthe chemical name:N-(5-((5-tert-butyloxazol-2-yl)methylthio)thiazol-2-yl)piperidine-4-carboxamide;and has the following structure:

In another embodiment, the CDK inhibitor is NC381. NC381 inhibits theactivity of cell-cycle CDKs, including CDK4. NC381 has the followingstructure:

In another embodiment, the CDK inhibitor is Milciclib. Milciclib is anorally bioavailable inhibitor of cyclin-dependent kinases (CDKs) andthropomyosin receptor kinase A (TRKA). Milciclib inhibits the activityof cell-cycle CDKs, including CDK1, CDK2, and CDK4. Milciclib has thechemical name:N,1,4,4-tetramethyl-8-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide;and has the following structure:

In another embodiment, the CDK inhibitor is ON123300. ON123300 inhibitsthe activity of cell-cycle CDKs, including CDK4. ON123300 has thechemical name: NH—(N—CH3piperazino)phenyl; and has the followingstructure:

In another embodiment, the CDK inhibitor is PD0332991/palbociclib.PD0332991/palbociclib inhibits the activity of CDKs, including CDK4 andCDK6, with IC50 of 11 nM and 16 nM, respectively. PD0332991/palbociclibhas the chemical name: Ethanesulfonic acid, 2-hydroxy-, compd. with6-acetyl-8-cyclopentyl-5-methyl-2-[[5-(1-piperazinyl)-2-pyridinyl]amino]pyrido[2,3-d]pyrimidin-7(8H)-one(1:1); and has the following structure:

In some embodiments, an anti-cancer agent is other than a CDK inhibitor.Exemplary anti-cancer agents that are not CDK inhibitors, include, e.g.,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), 5-azacytidine, 6-mercaptopurine,6-thioguanine, actinomycin D, amsacrine, bis-chloroethylnitrosurea,bleomycin, bryostatin-1, busulfan, carboplatin (Paraplatin®),chlorambucil, cisplatin (Platinol®), cetuximab (Erbitux®), colchicine,cyclophosphamide, cytarabine, cytosine arabinoside, dacarbazine,daunorubicin, daunomycin, dactinomycin, deoxycoformycin,diethylstilbestrol (DES), doxorubicin, etoposide (VP-16), epirubicin,esorubicin, fluorouracil (5-FU, Adrucil), gemcitabine,hexamethylmelamine, hydroxyprogesterone, hydroxyurea, idarubicin,ifosfamide, irinotecan, mafosfamide, melphalan, methotrexate (MTX),methylcyclohexylnitrosurea, mithramycin, mitomycin C, mitoxantrone,nitrogen mustards, paclitaxel (Taxol®), pentamethylmelamine, prednisone,procarbazine, tamoxifen, taxol, teniposide, testosterone, trimetrexate,topotecan, vincristine, and vinblastine.

In some embodiments, a patient is HPV+, and the patient is administeredan agent to treat the HPV infection, such as interferon or Famvir.

In other embodiments, the CDK inhibitor or other inhibitor isadministered in combination with a second therapeutic agent or adifferent therapeutic modality, e.g., an anti-cancer agent, and/or incombination with surgical and/or radiation procedures. In otherembodiments, the CDK inhibitor is administered in combination with asecond therapeutic agent or a different therapeutic modality, e.g., totreat a symptom of chemotherapy such as for treatment of nausea orheadache.

By “in combination with,” it is not intended to imply that the therapyor the therapeutic agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. The pharmaceutical compositions canbe administered concurrently with, prior to, or subsequent to, one ormore other additional therapies or therapeutic agents. In general, eachagent will be administered at a dose and/or on a time scheduledetermined for that agent. It will further be appreciated that theadditional therapeutic agent utilized in this combination can beadministered together in a single composition or administered separatelyin different compositions. The particular combination to employ in aregimen will take into account compatibility of the inventivepharmaceutical composition with the additional therapeutically activeagent and/or the desired therapeutic effect to be achieved.

For example, the second therapeutic agent can be a cytotoxic or acytostatic agent. Exemplary cytotoxic agents include antimicrotubuleagents, topoisomerase inhibitors, or taxanes, antimetabolites, mitoticinhibitors, alkylating agents, intercalating agents, agents capable ofinterfering with a signal transduction pathway, agents that promoteapoptosis and radiation. In yet other embodiments, the methods can beused in combination with immunodulatory agents, e.g., IL-1, 2, 4, 6, or12, or interferon α or γ, or immune cell growth factors such as GM-CSF.

In certain embodiments, the CDK inhibitor, e.g., the CDK 4/6 inhibitor,is administered in combination with another agent, e.g., one or moreanti-cancer agents. Exemplary anti-cancer agent combinations with theCDK include, but are not limited to, a PI3K inhibitor (e.g., a PI3Kinhibitor as described herein and in, e.g., WO 2013/006532); an mTORinhibitor (e.g., an mTOR inhibitor as described herein and in, e.g., WO2011/130232); and a fibroblast growth factor receptor inhibitor, e.g., apan-FGFR inhibitor or an FGFR3 inhibitor as described in, e.g., WO2006/000420 and WO 2013/006368, incorporated herein by reference.

In some embodiments, the anti-cancer agent inhibits expression of anucleic acid encoding a mutant gene, such as a mutant gene describedherein. Examples of such antagonists include nucleic acid molecules, forexample, antisense molecules, ribozymes, RNAi, triple helix moleculesthat hybridize to a nucleic acid encoding a cell cycle gene, or atranscription regulatory region, and block or reduce mRNA expression ofa mutant cell cycle gene.

In some embodiments, the anti-cancer agent is a nucleic acid molecule,such as an antisense molecule, a ribozyme, an siRNA, or a triple helixmolecule that hybridizes to a nucleic acid encoding a mutant gene, suchas a mutant cell-cycle gene, or a transcription regulatory region, andblocks or reduces mRNA expression of the mutant gene.

An “antisense” nucleic acid can include a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. The antisense nucleic acid can becomplementary to an entire mutant gene coding strand, or to only aportion thereof. For example, the antisense nucleic acid can becomplementary to the sequence in the ligand-binding domain that carriesthe mutation, e.g., can be complementary to the fusion junction in theligand-binding domain created by the six-nucleotide deletion describedherein. In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding a mutant gene (e.g., the 5′ and 3′ untranslatedregions). Anti-sense agents can include, for example, from about 8 toabout 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g.,about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases.Anti-sense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA can interfere withone or more of the normal functions of mRNA. The functions of mRNA to beinterfered with include all key functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding the mutant gene. The complementary region can extend forbetween about 8 to about 80 nucleobases. The compounds can include oneor more modified nucleobases. Modified nucleobases are known in the art.Descriptions of modified nucleic acid agents are also available. See,e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and U.S. Pat. No. 5,093,246;Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D. A.Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59;Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15.

siRNAs are small double stranded RNAs (dsRNAs) that optionally includeoverhangs. For example, the duplex region of an siRNA is about 18 to 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotidesin length. Typically, the siRNA sequences are exactly complementary tothe target mRNA. dsRNAs and siRNAs in particular can be used to silencegene expression in mammalian cells (e.g., human cells). siRNAs alsoinclude short hairpin RNAs (shRNAs) with 29-base-pair stems and2-nucleotide 3′ overhangs. See, e.g., Clemens et al. (2000) Proc. Natl.Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et al.(2002) Proc. Natl. Acad. Sci. USA 99:9942-9947; Siolas et al. (2005),Nat. Biotechnol. 23(2):227-31; 20040086884; U.S. 20030166282;20030143204; 20040038278; and 20030224432.

A ribozyme having specificity for a mutant gene-encoding nucleic acidcan include one or more sequences complementary to the nucleotidesequence of a mutant gene cDNA disclosed herein, and a sequence havingknown catalytic sequence responsible for mRNA cleavage (see U.S. Pat.No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a mutant gene-encoding mRNA.See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S.Pat. No. 5,116,742. Alternatively, a mutant gene mRNA can be used toselect a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993)Science 261:1411-1418.

Mutant gene expression can also be inhibited by targeting nucleotidesequences complementary to the regulatory region of the mutant gene toform triple helical structures that prevent transcription of the mutantgene in target cells. See generally, Helene, C. (1991) Anticancer DrugDes. 6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; andMaher, L. J. (1992) Bioassays 14:807-15. The potential sequences thatcan be targeted for triple helix formation can be increased by creatinga so-called “switchback” nucleic acid molecules. Switchback moleculesare synthesized in an alternating 5′-3′, 3′-5′ manner, such that theybase pair with first one strand of a duplex and then the other,eliminating the necessity for a sizeable stretch of either purines orpyrimidines to be present on one strand of a duplex.

In some embodiments, an anti-cancer agent is a peptide nucleic acid(PNA). For example, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, for example,inducing transcription or translation arrest or inhibiting replication.PNAs of mutant nucleic acid molecules can also be used in the analysisof single base pair mutations in a gene (e.g., by PNA-directed PCRclamping); as ‘artificial restriction enzymes’ when used in combinationwith other enzymes (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)).

In other embodiments, an anti-cancer agent that is a nucleic acid mayinclude other appended groups such as peptides (e.g., for targeting hostcell receptors in vivo), or agents facilitating transport across thecell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci.USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; WO88/09810) or the blood-brain barrier (see, e.g., WO89/10134). In addition, oligonucleotides can be modified withhybridization-triggered cleavage agents (see, e.g., Krol et al. (1988)Bio-Techniques 6:958-976) or intercalating agents (See, e.g., Zon (1988)Pharm. Res. 5:539-549). To this end, the anti-cancer agent may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent.

Methods for Detecting Viral and Mutant Nucleic Acids and Polypeptides

In another aspect, the invention features a method of determining thepresence of an HPV nucleic acid, and/or a mutant gene, e.g., a mutantgene as described herein. In one embodiment, the HPV nucleic acid and/orthe mutant gene (e.g., a mutant cell cycle gene) is identified bydetection of an HPV nucleic acid molecule or polypeptide, and/or amutant nucleic acid molecule or polypeptide. The method includesdetecting whether a mutant nucleic acid molecule or polypeptide ispresent in a cell (e.g., a circulating cell), a tissue (e.g., a tumor),or a sample, e.g., a tumor sample, from a subject. In one embodiment,the sample is a nucleic acid sample. In one embodiment, the nucleic acidsample comprises DNA, e.g., genomic DNA or cDNA, or RNA, e.g., mRNA. Inother embodiments, the sample is a protein sample.

In one embodiment, the sample is, or has been, classified asnon-malignant using other diagnostic techniques, e.g.,immunohistochemistry.

In one embodiment, the sample is, or has been, classified as malignantusing other diagnostic techniques, e.g., immunohistochemistry.

In one embodiment, the sample is acquired from a subject (e.g., asubject, e.g., a patient, having or at risk of having a cancer, e.g., apatient), or alternatively, the method further includes acquiring asample from the subject. The sample can be chosen from one or more of:tissue, e.g., cancerous tissue (e.g., a tissue biopsy), whole blood,serum, plasma, buccal scrape, sputum, saliva, cerebrospinal fluid,urine, stool, circulating tumor cells, circulating nucleic acids, orbone marrow. In certain embodiments, the sample is a tissue (e.g., atumor biopsy), a circulating tumor cell or nucleic acid.

In embodiments, the tumor is from a cancer described herein, e.g., headand neck cancer, such as an HNSCC. In other embodiments, the cancer is ametastasis.

In one embodiment, the subject is at risk of having, or has a squamouscell carcinoma of the head and neck.

In other embodiments, the HPV and/or mutant gene is detected in anucleic acid molecule by a method chosen from one or more of: nucleicacid hybridization assay, amplification-based assays (e.g., polymerasechain reaction (PCR)), PCR-RFLP assay, real-time PCR, sequencing,screening analysis (including metaphase cytogenetic analysis by standardkaryotype methods, FISH (e.g., break away FISH), spectral karyotyping orMFISH, comparative genomic hybridization), in situ hybridization, SSP,HPLC or mass-spectrometric genotyping.

In one embodiment, the method includes: contacting a nucleic acidsample, e.g., a genomic DNA sample (e.g., a chromosomal sample or afractionated, enriched or otherwise pre-treated sample) or a geneproduct (mRNA, cDNA), obtained from the subject, with a nucleic acidfragment (e.g., a probe or primer as described herein (e.g., anexon-specific probe or primer) under conditions suitable forhybridization, and determining the presence or absence of the mutantnucleic acid molecule. The method can, optionally, include enriching asample for the gene or gene product.

In a related aspect, a method for determining the presence of an HPVand/or mutant nucleic acid molecule is provided. The method includes:acquiring a sequence for a position in a nucleic acid molecule, e.g., bysequencing at least one nucleotide of the nucleic acid molecule (e.g.,sequencing at least one nucleotide in the nucleic acid molecule thatcomprises the mutant gene), thereby determining that the mutant gene ispresent in the nucleic acid molecule. Optionally, the sequence acquiredis compared to a reference sequence, or a wild type reference sequence.In one embodiment, the nucleic acid molecule is from a cell (e.g., acirculating cell), a tissue (e.g., a tumor), or any sample from asubject (e.g., blood or plasma sample). In other embodiments, thenucleic acid molecule from a tumor sample (e.g., a tumor or cancersample) is sequenced. In one embodiment, the sequence is determined by anext generation sequencing method. The method further can furtherinclude acquiring, e.g., directly or indirectly acquiring, a sample,e.g., a tumor or cancer sample, from a subject (e.g., a patient). Incertain embodiments, the cancer is an HNSCC.

In another aspect, the invention features a method of analyzing a tumoror a circulating tumor cell. The method includes acquiring a nucleicacid sample from the tumor or the circulating cell; and sequencing,e.g., by a next generation sequencing method, a nucleic acid molecule,e.g., a nucleic acid molecule that includes a mutant gene describedherein. In one embodiment the invention features a method of analyzing ametastasis, e.g., in a tissue separate from the site of the primarytumor.

In one embodiment, probes or primers can be designed to detect amutation in a cell cycle gene, such as a CDKN2A or CDKN2B or CCND1. Inother embodiment, probes or primers can be design to detect an HPVstrain.

In one embodiment, amplification-based assays can be used to measure thepresence or absence of a gene, or copy number. In suchamplification-based assays, the nucleic acid sequences act as a templatein an amplification reaction (e.g., Polymerase Chain Reaction (PCR)). Ina quantitative amplification, the amount of amplification product willbe proportional to the amount of template in the original sample.Comparison to appropriate controls, e.g., healthy tissue, provides ameasure of the copy number.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that can be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis, et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR can also be used in the methods of theinvention. In fluorogenic quantitative PCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan® and SYBR® green.

In one embodiment, a TaqMan® assay is used to identify a mutation in agene, e.g., a mutation as described herein, such as by utilizing a probethat binds specifically to the mutation, and a control probe that bindsto the wildtype sequence, and probes that bind outside of the mutatedsequence for PCR amplification.

Other suitable amplification methods include, but are not limited to,ligase chain reaction

(LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al.(1988) Science 241:1077, and Barringer et al. (1990) Gene 89: 117),transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci.USA 86: 1173), self-sustained sequence replication (Guatelli, et al.(1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapterPCR, etc.

Chromosomal probes are typically about 50 to about 10⁵ nucleotides inlength. Longer probes typically comprise smaller fragments of about 100to about 500 nucleotides in length. Probes that hybridize withcentromeric DNA and locus-specific DNA are available commercially, forexample, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc.(Eugene, Oreg.) or from Cytocell (Oxfordshire, UK). Alternatively,probes can be made non-commercially from chromosomal or genomic DNAthrough standard techniques. For example, sources of DNA that can beused include genomic DNA, cloned DNA sequences, somatic cell hybridsthat contain one, or a part of one, chromosome (e.g., human chromsome)along with the normal chromosome complement of the host, and chromosomespurified by flow cytometry or microdis section. The region of interestcan be isolated through cloning, or by site-specific amplification viathe polymerase chain reaction (PCR). See, for example, Nath and Johnson,Biotechnic Histochem., 1998, 73(1):6-22, Wheeless et al., Cytometry1994, 17:319-326, and U.S. Pat. No. 5,491,224.

Additional exemplary methods include, traditional “direct probe” methodssuch as Southern blots or in situ hybridization (e.g., fluorescence insitu hybridization (FISH) and FISH plus SKY), and “comparative probe”methods such as comparative genomic hybridization (CGH), e.g.,cDNA-based or oligonucleotide-based CGH, can be used. The methods can beused in a wide variety of formats including, but not limited to,substrate (e.g., membrane or glass) bound methods or array-basedapproaches.

Additional Protocols for FISH Detection are Described Below.

The probes to be used hybridize to a specific region of a chromosome todetermine whether a cytogenetic abnormality is present in this region.One type of cytogenetic abnormality is a deletion. Although deletionscan be of one or more entire chromosomes, deletions normally involveloss of part of one or more chromosomes. If the entire region of achromosome that is contained in a probe is deleted from a cell,hybridization of that probe to the DNA from the cell will normally notoccur and no signal will be present on that chromosome. If the region ofa chromosome that is partially contained within a probe is deleted froma cell, hybridization of that probe to the DNA from the cell can stilloccur, but less of a signal can be present. For example, the loss of asignal is compared to probe hybridization to DNA from control cells thatdo not contain the genetic abnormalities which the probes are intendedto detect. In some embodiments, at least 1, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, ormore cells are enumerated for presence of the cytogenetic abnormality.

Cytogenetic abnormalities to be detected can include, but are notlimited to, non-reciprocal translocations, intra-chromosomal inversions,point mutations, deletions, gene copy number changes, gene expressionlevel changes, and germ line mutations. In particular, one type ofcytogenetic abnormality is a duplication. Duplications can be of entirechromosomes, or of regions smaller than an entire chromosome. If theregion of a chromosome that is contained in a probe is duplicated in acell, hybridization of that probe to the DNA from the cell will normallyproduce at least one additional signal as compared to the number ofsignals present in control cells with no abnormality of the chromosomalregion contained in the probe.

Chromosomal probes are labeled so that the chromosomal region to whichthey hybridize can be detected. Probes typically are directly labeledwith a fluorophore, an organic molecule that fluoresces after absorbinglight of lower wavelength/higher energy. The fluorophore allows theprobe to be visualized without a secondary detection molecule. Aftercovalently attaching a fluorophore to a nucleotide, the nucleotide canbe directly incorporated into the probe with standard techniques such asnick translation, random priming, and PCR labeling. Alternatively,deoxycytidine nucleotides within the probe can be transaminated with alinker. The fluorophore then is covalently attached to the transaminateddeoxycytidine nucleotides. See, U.S. Pat. No. 5,491,224.

U.S. Pat. No. 5,491,224 describes probe labeling as a number of thecytosine residues having a fluorescent label covalently bonded thereto.The number of fluorescently labeled cytosine bases is sufficient togenerate a detectable fluorescent signal while the individual so labeledDNA segments essentially retain their specific complementary binding(hybridizing) properties with respect to the chromosome or chromosomeregion to be detected. Such probes are made by taking the unlabeled DNAprobe segment, transaminating with a linking group a number ofdeoxycytidine nucleotides in the segment, covalently bonding afluorescent label to at least a portion of the transaminateddeoxycytidine bases.

Probes can also be labeled by nick translation, random primer labelingor PCR labeling. Labeling is done using either fluorescent (direct)- orhaptene (indirect)-labeled nucleotides. Representative, non-limitingexamples of labels include: AMCA-6-dUTP, CascadeBlue-4-dUTP,Fluorescein-12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP,Cy5-dUTP, Biotin(BIO)-11-dUTP, Digoxygenin(DIG)-11-dUTP or Dinitrophenyl(DNP)-11-dUTP.

Probes also can be indirectly labeled with biotin or digoxygenin, orlabeled with radioactive isotopes such as ³²P and .³H, althoughsecondary detection molecules or further processing then is required tovisualize the probes. For example, a probe labeled with biotin can bedetected by avidin conjugated to a detectable marker. For example,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Enzymatic markers can be detectedin standard colorimetric reactions using a substrate and/or a catalystfor the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Probes can also be prepared such that a fluorescent or other label isnot part of the DNA before or during the hybridization, and is addedafter hybridization to detect the probe hybridized to a chromosome. Forexample, probes can be used that have antigenic molecules incorporatedinto the DNA. After hybridization, these antigenic molecules aredetected using specific antibodies reactive with the antigenicmolecules. Such antibodies can themselves incorporate a fluorochrome, orcan be detected using a second antibody with a bound fluorochrome.

However treated or modified, the probe DNA is commonly purified in orderto remove unreacted, residual products (e.g., fluorochrome molecules notincorporated into the DNA) before use in hybridization.

Prior to hybridization, chromosomal probes are denatured according tomethods well known in the art. Probes can be hybridized or annealed tothe chromosomal DNA under hybridizing conditions. “Hybridizingconditions” are conditions that facilitate annealing between a probe andtarget chromosomal DNA. Since annealing of different probes will varydepending on probe length, base concentration and the like, annealing isfacilitated by varying probe concentration, hybridization temperature,salt concentration and other factors well known in the art.

Hybridization conditions are facilitated by varying the concentrations,base compositions, complexities, and lengths of the probes, as well assalt concentrations, temperatures, and length of incubation. Forexample, in situ hybridizations are typically performed in hybridizationbuffer containing 1× to 2×SSC, 50% to 65% formamide and blocking DNA tosuppress non-specific hybridization. In general, hybridizationconditions, as described above, include temperatures of about 25° C. toabout 55° C., and incubation lengths of about 0.5 hours to about 96hours.

Non-specific binding of chromosomal probes to DNA outside of the targetregion can be removed by a series of washes. Temperature andconcentration of salt in each wash are varied to control stringency ofthe washes. For example, for high stringency conditions, washes can becarried out at about 65° C. to about 80° C., using 0.2× to about 2×SSC,and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40(NP40). Stringency can be lowered by decreasing the temperature of thewashes or by increasing the concentration of salt in the washes. In someapplications it is necessary to block the hybridization capacity ofrepetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-I DNA is used to block non-specific hybridization. Afterwashing, the slide is allowed to drain and air dry, then mountingmedium, a counterstain such as DAPI, and a coverslip are applied to theslide. Slides can be viewed immediately or stored at −20° C. beforeexamination.

For fluorescent probes used in fluorescence in situ hybridization (FISH)techniques, fluorescence can be viewed with a fluorescence microscopeequipped with an appropriate filter for each fluorophore, or by usingdual or triple band-pass filter sets to observe multiple fluorophores.See, for example, U.S. Pat. No. 5,776,688. Alternatively, techniquessuch as flow cytometry can be used to examine the hybridization patternof the chromosomal probes.

In CGH methods, a first collection of nucleic acids (e.g., from asample, e.g., a possible tumor) is labeled with a first label, while asecond collection of nucleic acids (e.g., a control, e.g., from ahealthy cell/tissue) is labeled with a second label. The ratio ofhybridization of the nucleic acids is determined by the ratio of the two(first and second) labels binding to each fiber in the array. Wherethere are chromosomal deletions or multiplications, differences in theratio of the signals from the two labels will be detected and the ratiowill provide a measure of the copy number. Array-based CGH can also beperformed with single-color labeling (as opposed to labeling the controland the possible tumor sample with two different dyes and mixing themprior to hybridization, which will yield a ratio due to competitivehybridization of probes on the arrays). In single color CGH, the controlis labeled and hybridized to one array and absolute signals are read,and the possible tumor sample is labeled and hybridized to a secondarray (with identical content) and absolute signals are read. Copynumber difference is calculated based on absolute signals from the twoarrays.

Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430,402; Methods in Molecular Biology, Vol. 33: In situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In oneembodiment, the hybridization protocol of Pinkel, et al. (1998) NatureGenetics 20: 207-211, or of Kallioniemi (1992) Proc. Natl Acad Sci USA89:5321-5325 (1992) is used. Array-based CGH is described in U.S. Pat.No. 6,455,258, the contents of each of which are incorporated herein byreference.

In yet another embodiment, the level (e.g., expression level) oractivity of the mutant gene is evaluated. For example, the level (e.g.,expression level) or activity of the mutant gene (e.g., mRNA orpolypeptide) is detected and (optionally) compared to a pre-determinedvalue, e.g., a reference value (e.g., a control sample).

In yet other embodiments, a viral or mutant polypeptide is detected. Themethod includes: contacting a protein sample with a reagent whichspecifically binds to a viral polypeptide or mutant polypeptide; anddetecting the formation of a complex of the polypeptide and the reagent.In one embodiment, the reagent is labeled with a detectable group tofacilitate detection of the bound and unbound reagent. In oneembodiment, the reagent is an antibody molecule, e.g., is selected fromthe group consisting of an antibody, and antibody derivative, and anantibody fragment.

The activity or level of a viral polypeptide or mutant polypeptide canalso be detected and/or quantified by detecting or quantifying theexpressed polypeptide. The polypeptide can be detected and quantified byany of a number of means known to those of skill in the art. These caninclude analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,or various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, immunohistochemistry (IHC)and the like. A skilled artisan can adapt known protein/antibodydetection methods.

Another agent for detecting a viral polypeptide or mutant polypeptide isan antibody molecule capable of binding to the polypeptide, e.g., anantibody with a detectable label. Techniques for generating antibodiesare described herein. The term “labeled,” with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin.

In another embodiment, the antibody is labeled, e.g., a radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. Inanother embodiment, an antibody derivative (e.g., an antibody conjugatedwith a substrate or with the protein or ligand of a protein-ligand pair(e.g., biotin-streptavidin)), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a mutant protein, is used.

Mutant polypeptides from cells can be isolated using techniques that areknown to those of skill in the art. The protein isolation methodsemployed can, for example, be such as those described in Harlow and Lane(Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

Means of detecting proteins using electrophoretic techniques are wellknown to those of skill in the art (see generally, R. Scopes (1982)Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc., N.Y.).

In another embodiment, Western blot (immunoblot) analysis is used todetect and quantify the presence of a polypeptide in the sample.

In another embodiment, the polypeptide is detected using an immunoassay.As used herein, an immunoassay is an assay that utilizes an antibody tospecifically bind to the analyte. The immunoassay is thus characterizedby detection of specific binding of a polypeptide to an anti-antibody asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte.

The mutant polypeptide is detected and/or quantified using any of anumber of immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

HPV Molecule and/or Mutant Gene Expression Level

In certain embodiments, HPV molecule and/or mutant gene expressionlevels (e.g., mutant cell cycle gene expression levels) can also beassayed. Gene expression can be assessed by any of a wide variety ofmethods for detecting expression of a transcribed molecule or protein.Non-limiting examples of such methods include immunological methods fordetection of secreted, cell-surface, cytoplasmic, or nuclear proteins,protein purification methods, protein function or activity assays,nucleic acid hybridization methods, nucleic acid reverse transcriptionmethods, and nucleic acid amplification methods.

In certain embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g., mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. Mutant gene expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

Methods of detecting and/or quantifying the HPV molecule and/or mutantgene transcript (mRNA or cDNA made therefrom) using nucleic acidhybridization techniques are known to those of skill in the art (seeSambrook et al. supra). For example, one method for evaluating thepresence, absence, or quantity of cDNA involves a Southern transfer asdescribed above. Briefly, the mRNA is isolated (e.g., using an acidguanidinium-phenol-chloroform extraction method, Sambrook et al. supra.)and reverse transcribed to produce cDNA. The cDNA is then optionallydigested and run on a gel in buffer and transferred to membranes.Hybridization is then carried out using the nucleic acid probes specificfor the mutant cDNA, e.g., using the probes and primers describedherein.

In other embodiments, HPV molecule and/or mutant gene expression isassessed by preparing genomic DNA or mRNA/cDNA (i.e., a transcribedpolynucleotide) from cells in a subject sample, and by hybridizing thegenomic DNA or mRNA/cDNA with a reference polynucleotide which is acomplement of a polynucleotide comprising the mutant gene, and fragmentsthereof. cDNA can, optionally, be amplified using any of a variety ofpolymerase chain reaction methods prior to hybridization with thereference polynucleotide. Expression of a mutant gene can likewise bedetected using quantitative PCR (QPCR) to assess the level of mutantgene expression.

Detection of HPV and/or Mutant Polypeptides

In another aspect, the invention features a mutant polypeptide (e.g., apurified mutant polypeptide), a biologically active or antigenicfragment thereof, as well as reagents (e.g., antibody molecules thatbind to a mutant polypeptide), methods for modulating a mutantpolypeptide activity and detection of a mutant polypeptide, e.g., amutant cell-cycle polypeptide.

In one embodiment, the mutant polypeptide has at least one biologicalactivity, e.g., a role in cell-cycle regulation. In one embodiment, atleast one biological activity of a mutant polypeptide is reduced orinhibited by an anti-cancer agent, e.g., cell cycle inhibitor, such as aCDK inhibitor, such as an inhibitor of one or both of CDK4 or CDK6.

In other embodiments, the mutant polypeptide includes a fragment of amutant polypeptide containing a mutation described herein.

In yet other embodiments, the mutant polypeptide is encoded by a nucleicacid molecule described herein.

In another embodiment, the mutant polypeptide or fragment thereof is apeptide, e.g., an immunogenic peptide or protein that contains amutation described herein. Such immunogenic peptides or proteins can beused to raise antibodies specific to the mutant protein. In otherembodiments, such immunogenic peptides or proteins can be used forvaccine preparation. The vaccine preparation can include othercomponents, e.g., an adjuvant.

In another aspect, the invention features antibody molecules that bindto a mutant polypeptide or fragment described herein. In embodiments theantibody can distinguish a wild-type from a mutant polypeptide.

In one aspect, the invention features a polypeptide comprising amutation identified in Table 1.

Kits

In one aspect, the invention features, a kit, e.g., containing methodsfor determining whether a subject is HPV+ or HPV−, and to accordinglyidentify an appropriate therapeutic agent to treat the a cancer, e.g., aHNSCC, in the subject. For example, in some embodiments the kit containsprimers, such as for PCR, or one or more probes for detecting an HPV ina sample from the subject. In other embodiment, the kit contains anantibody for detecting an HPV protein in a sample from the subject.

Optionally, the kit also contains an oligonucleotide having a mutationdescribed herein, e.g., an oligonucleotide that hybridizes specificallyto a mutation in a functional domain of a mutant gene, such as a mutantcell cycle gene. Optionally, the kit can also contain an oligonucleotidethat is the wildtype counterpart of the mutant oligonucleotide.

A kit featured in the invention can include a carrier, e.g., a meansbeing compartmentalized to receive in close confinement one or morecontainer means. In one embodiment the container contains anoligonucleotide, e.g., a primer or probe as described above. Thecomponents of the kit are useful, for example, to identify a mutation ina tumor sample in a patient. The probe or primer of the kit can be usedin any sequencing or nucleotide detection assay known in the art, e.g.,a sequencing assay, e.g., an NGS method, RT-PCR, or in situhybridization.

A kit featured in the invention can include, e.g., assay positive andnegative controls, nucleotides, enzymes (e.g., RNA or DNA polymerase orligase), solvents or buffers, a stabilizer, a preservative, a secondaryantibody, e.g., an anti-HRP antibody (IgG) and a detection reagent.

An oligonucleotide can be provided in any form, e.g., liquid, dried,semi-dried, or lyophilized, or in a form for storage in a frozencondition.

Typically, an oligonucleotide, and other components in a kit areprovided in a form that is sterile. When an oligonucleotide, e.g., anoligonucleotide that contains a mutation in a gene, e.g., a mutation ina functional domain of a gene as described herein, or an oligonucleotidecomplementary to a mutation, is provided in a liquid solution, theliquid solution generally is an aqueous solution, e.g., a sterileaqueous solution. When the oligonucleotide is provided as a dried form,reconstitution generally is accomplished by the addition of a suitablesolvent. The solvent, e.g., sterile buffer, can optionally be providedin the kit.

The kit can include one or more containers for the compositioncontaining an oligonucleotide in a concentration suitable for use in theassay or with instructions for dilution for use in the assay. In someembodiments, the kit contains separate containers, dividers orcompartments for the oligonucleotide and assay components, and theinformational material. For example, the oligonucleotides can becontained in a bottle or vial, and the informational material can becontained in a plastic sleeve or packet. In other embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, an oligonucleotide composition is contained in abottle or vial that has attached thereto the informational material inthe form of a label. In some embodiments, the kit includes a plurality(e.g., a pack) of individual containers, each containing one or moreunit forms (e.g., for use with one assay) of an oligonucleotide. Forexample, the kit includes a plurality of ampoules, foil packets, orblister packs, each containing a single unit of oligonucleotide for usein a sequencing or detecting a mutation in a tumor sample. Thecontainers of the kits can be air tight and/or waterproof. The containercan be labeled for use.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds specifically to an HPVprotein, or a mutant polypeptide associated with the HNSCC, such as apolypeptide that functions in the cell cycle; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

In one embodiment, the kit can include informational material forperforming and interpreting the sequencing or diagnostic. In anotherembodiment, the kit can provide guidance as to where to report theresults of the assay, e.g., to a treatment center or healthcareprovider. The kit can include forms for reporting the results of asequencing or diagnostic assay described herein, and address and contactinformation regarding where to send such forms or other relatedinformation; or a URL (Uniform Resource Locator) address for reportingthe results in an online database or an online application (e.g., anapp). In another embodiment, the informational material can includeguidance regarding whether a patient should receive treatment with aparticular chemotherapeutic drug, depending on the results of the assay.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawings, and/or photographs,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about the sequencingor diagnostic assay and/or its use in the methods described herein. Theinformational material can also be provided in any combination offormats.

In some embodiments, a biological sample is provided to an assayprovider, e.g., a service provider (such as a third party facility) or ahealthcare provider, who evaluates the sample in an assay and provides aread out. For example, in one embodiment, an assay provider receives abiological sample from a subject, such as a blood or tissue sample,e.g., a biopsy sample, and evaluates the sample using an assay describedherein, e.g., a sequencing assay or in situ hybridization assay, anddetermines that the sample contains a nucleic acid containing a mutationdescribed in Table 1. The assay provider, e.g., a service provider orhealthcare provider, can then conclude that the subject is, or is not, acandidate for a particular drug or a particular cancer treatmentregimen.

The assay provider can provide the results of the evaluation, andoptionally, conclusions regarding one or more of diagnosis, prognosis,or appropriate therapy options to, for example, a healthcare provider,or patient, or an insurance company, in any suitable format, such as bymail or electronically, or through an online database. The informationcollected and provided by the assay provider can be stored in adatabase.

The invention is further illustrated by the following example, whichshould not be construed as further limiting.

Systems or Devices

In another aspect, the invention features a system or a device (e.g., asequencing device) for producing a report, e.g., a report for recordingthe presence or absence of HPV in a patient. The system or device caninclude a component for containing a sample (e.g., a blood or serumsample, or tumor sample from a patient); a detection component capableof identifying the presence or absence of a HPV, e.g., a sequence of anHPV gene or gene fragment as described herein; and a means foroutputting a report, e.g., a report as described herein. For example,the detection component can include a probe or primer for detecting asequence of an HPV, such as by a sequencing method, such as by PCR.

In another aspect, the invention features a system or a device (e.g., asequencing device) for producing a report, e.g., a genotype report. Thesystem or device can include a component for containing a sample (e.g.,a tumor nucleic acid or polypeptide); a detection component capable ofidentifying the presence or absence of a mutant gene, e.g., a mutantcell cycle gene as described herein; and a means for outputting areport, e.g., a report as described herein.

In one embodiment, the component for containing a tumor sample isconfigured in a way to contain or hold the sample, e.g., a tumor nucleicacid or polypeptide sample.

In another embodiment, the detection component produces and/or analyzesa signal according to the presence or absence of the mutant gene in thesample.

In another embodiment, the means for outputting a report provides asystem for annotating the association of the detected mutant gene to thesample. The report can include, e.g., the identification of nucleotidevalues, the indication of presence or absence of a mutant gene asdescribed herein, or sequence, and whether the sample was obtained froman HPV+ or HPV− patient. In one embodiment, a report is generated, suchas in paper or electronic form, which identifies the presence or absenceof an alteration described herein, and optionally includes an identifierfor the patient from which the sequence was obtained.

The report can also include information on the role of a sequence, e.g.,a mutant cell cycle gene as described herein, or wild-type sequence, indisease. Such information can include information on prognosis,resistance, or potential or suggested therapeutic options. The reportcan include information on the likely effectiveness of a therapeuticoption, the acceptability of a therapeutic option, or the advisabilityof applying the therapeutic option to a patient, e.g., a patient havinga sequence, alteration or mutation identified in the test, and inembodiments, identified in the report.

Nucleic Acid Detection and Capturing Reagents

The invention also features a nucleic acid molecule, e.g., nucleic acidfragment, suitable as probe, primer, bait or library member thatincludes, flanks, hybridizes to, and which are useful for identifying,or are otherwise based on, the mutants described herein. In certainembodiments, the probe, primer or bait molecule is an oligonucleotidethat allows capture, detection or isolation of a mutant nucleic acidmolecule, e.g., a mutant cell cycle gene or gene of the PI3K genedescribed herein.

In one embodiment, the nucleic acid fragment is a probe or primer thatincludes an oligonucleotide between about 5 and 25, e.g., between 10 and20, or 10 and 15 nucleotides in length. In other embodiments, thenucleic acid fragment is a bait that includes an oligonucleotide betweenabout 100 to 300 nucleotides, 130 and 230 nucleotides, or 150 and 200nucleotides, in length.

In one embodiment, the nucleic acid fragment can be used to identify orcapture, e.g., by hybridization, a mutant cell cycle gene. For example,the nucleic acid fragment can be a probe, a primer, or a bait, for usein identifying or capturing, e.g., by hybridization, a mutant cell cyclegene described herein.

The probes or primers described herein can be used, for example, forFISH detection or PCR amplification. In one exemplary embodiment wheredetection is based on PCR, amplification of an cell cycle gene mutation,can be performed using a primer or a primer pair, e.g., for amplifying amutant sequence described herein. In one embodiment, a pair of isolatedoligonucleotide primers can amplify a region containing or adjacent toan mutation.

In another embodiment, the nucleic acid fragments can be used toidentify, e.g., by hybridization, a mutant gene.

In other embodiments, the nucleic acid fragment includes a bait thatcomprises a nucleotide sequence that hybridizes to a mutant nucleic acidmolecule described herein, and thereby allows the capture or isolationof said nucleic acid molecule. In one embodiment, a bait is suitable forsolution phase hybridization. In other embodiments, a bait includes abinding entity, e.g., an affinity tag, that allows capture andseparation, e.g., by binding to a binding entity, of a hybrid formed bya bait and a nucleic acid hybridized to the bait.

In other embodiments, the nucleic acid fragment includes a librarymember comprising a mutant nucleic acid molecule described herein.

The nucleic acid fragment can be detectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label, or caninclude an affinity tag; a tag, or identifier (e.g., an adaptor, barcodeor other sequence identifier).

One aspect of the invention pertains to isolated nucleic acid moleculesthat include a mutation, including nucleic acids which encode apolypeptide that contains a mutation in a structural domain, or aportion of such a polypeptide. The nucleic acid can include a cell cyclegene, such as a CDKN2A, CDKN2B or CCND1 gene. The nucleic acid moleculesinclude those nucleic acid molecules which reside in genomic regionsidentified herein. As used herein, the term “nucleic acid molecule”includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded; in certain embodiments the nucleic acid molecule isdouble-stranded DNA.

Isolated nucleic acid molecules also include nucleic acid moleculessufficient for use as hybridization probes or primers to identifynucleic acid molecules that contain a mutation in a gene, e.g., a cellcycle gene, e.g., nucleic acid molecules suitable for use as PCR primersfor the amplification or mutation of nucleic acid molecules.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. In certain embodiments, an “isolated” nucleicacid molecule is free of sequences (such as protein-encoding sequences)which naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

The language “substantially free of other cellular material or culturemedium” includes preparations of nucleic acid molecule in which themolecule is separated from cellular components of the cells from whichit is isolated or recombinantly produced. Thus, nucleic acid moleculethat is substantially free of cellular material includes preparations ofnucleic acid molecule having less than about 30%, less than about 20%,less than about 10%, or less than about 5% (by dry weight) of othercellular material or culture medium.

A nucleic acid molecule containing a mutation in a cell cycle gene canbe isolated using standard molecular biology techniques and the sequenceinformation in the database records described herein. Using all or aportion of such nucleic acid sequences, mutant nucleic acid moleculescan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook et al., ed., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Probes based on the sequence of a mutant nucleic acid molecule can beused to detect transcripts or genomic sequences corresponding to one ormore markers featured in the invention. The probe comprises a labelgroup attached thereto, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as part of atest kit for identifying cells or tissues which express a mutantprotein, such as by measuring levels of a nucleic acid molecule encodingthe protein in a sample of cells from a subject, e.g., detecting mRNAlevels or determining whether a gene encoding the protein has beenmutated or deleted.

Probes featured in the invention include those that will specificallyhybridize to a gene sequence described in the Table 1, e.g., a cellcycle gene, such as CDKN2A or CDKN2B, or

CCND1. In some embodiments, probes featured in the invention willspecifically hybridize to a gene rearrangement described in Table 4.Typically these probes are 12 to 20, e.g., 17 to 20 nucleotides inlength (longer for large insertions) and have the nucleotide sequencecorresponding to the region of the mutations at their respectivenucleotide locations on the gene sequence. Such molecules can be labeledaccording to any technique known in the art, such as with radiolabels,fluorescent labels, enzymatic labels, sequence tags, biotin, otherligands, etc. As used herein, a probe that “specifically hybridizes” toa mutant gene sequence will hybridize under high stringency conditions.

A probe will typically contain one or more of the specific mutationsdescribed herein. Typically, a nucleic acid probe will encompass onlyone mutation. Such molecules may be labeled and can be used asallele-specific probes to detect the mutation of interest.

The term “primer” as used herein refers to a sequence comprising two ormore deoxyribonucleotides or ribonucleotides, e.g., more than three, andmore than eight, or at least 20 nucleotides of a gene described in Table1 or Table 4, where the sequence corresponds to a sequence flanking oneof the mutations or a wildtype sequence of a gene identified in Table 1or Table 4, e.g., a CDKN2A or CDKN2B gene or a CCND1 gene. Primers maybe used to initiate DNA synthesis via the PCR (polymerase chainreaction) or a sequencing method. Primers featured in the inventioninclude the sequences recited and complementary sequences which wouldanneal to the opposite DNA strand of the sample target. Since bothstrands of DNA are complementary and mirror images of each other, thesame segment of DNA will be amplified.

Primers can be used to sequence a nucleic acid, e.g., an isolatednucleic acid described herein, such as by an NGS method, or to amplify agene described in Table 1 or Table 4, such as by PCR. The primers canspecifically hybridize, for example, to the ends of the exons or to theintrons flanking the exons. The amplified segment can then be furtheranalyzed for the presence of the mutation such as by a sequencingmethod, or by a size separation technique such as by electrophoresis ona gel. The primers are useful in directing amplification of a targetpolynucleotide prior to sequencing. Such primers are useful in directingamplification of a target region that includes a mutation, e.g., priorto sequencing. The primer typically contains 12 to 20, or 17 to 20, ormore nucleotides, although a primer may contain fewer nucleotides.

A primer is typically single stranded, e.g., for use in sequencing oramplification methods, but may be double stranded. If double stranded,the primer may first be treated to separate its strands before beingused to prepare extension products. A primer must be sufficiently longto prime the synthesis of extension products in the presence of theinducing agent for polymerization. The exact length of primer willdepend on many factors, including applications (e.g., amplificationmethod), temperature, buffer, and nucleotide composition. A primertypically contains 12-20 or more nucleotides, although a primer maycontain fewer nucleotides.

Primers are typically designed to be “substantially” complementary toeach strand of a genomic locus to be amplified. Thus, the primers mustbe sufficiently complementary to specifically hybridize with theirrespective strands under conditions which allow the agent forpolymerization to perform. In other words, the primers should havesufficient complementarity with the 5′ and 3′ sequences flanking themutation to hybridize therewith and permit amplification of the genomiclocus.

The term “substantially complementary to” or “substantially thesequence” refers to sequences that hybridize to the sequences providedunder stringent conditions and/or sequences having sufficient homologywith a sequence comprising a mutation, e.g., a point mutation or fusionjunction, or the wildtype counterpart sequence, such that the allelespecific oligonucleotides hybridize to the sequence. In one embodiment,a sequence is substantially complementary to a fusion junction createdby a deletion event or a translocation. “Substantially the same” as itrefers to oligonucleotide sequences also refers to the functionalability to hybridize or anneal with sufficient specificity todistinguish between the presence or absence of the mutation. This ismeasurable by the temperature of melting being sufficiently different topermit easy identification of whether the oligonucleotide is binding tothe normal or mutant gene sequence identified in the Example.

In one aspect, the invention features a primer or primer set foramplifying a nucleic acid comprising a mutation in a cell cycle gene.

Nucleic Acid Samples

A variety of tissue samples can be the source of the nucleic acidsamples used in the present methods, e.g., the source of the nucleicacid sample to assay for the presence of an HPV nucleic acid or amutation in a gene described herein, such as a gene or gene alterationdescribed in Table 1 or Table 4. Genomic or subgenomic DNA fragments canbe isolated from a subject's sample (e.g., a tumor sample, a normaladjacent tissue (NAT), a blood sample or any normal control)). Incertain embodiments, the tissue sample is preserved as a frozen sampleor as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE)tissue preparation. For example, the sample can be embedded in a matrix,e.g., an FFPE block or a frozen sample. The isolating step can includeflow-sorting of individual chromosomes; and/or micro-dissecting asubject's sample (e.g., a tumor sample, a NAT, a blood sample).

Protocols for DNA isolation from a tissue sample are known in the art.Additional methods to isolate nucleic acids (e.g., DNA) fromformaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE)tissues are disclosed, e.g., in Cronin M. et al., (2004) Am J Pathol.164(1):35-42; Masuda N. et al., (1999) Nucleic Acids Res.27(22):4436-4443; Specht K. et al., (2001) Am J Pathol. 158(2):419-429,Ambion RecoverAll™ Total Nucleic Acid Isolation Protocol (Ambion, Cat.No. AM1975, September 2008), and QIAamp® DNA FFPE Tissue Handbook(Qiagen, Cat. No. 37625, October 2007). RecoverAll™ Total Nucleic AcidIsolation Kit uses xylene at elevated temperatures to solubilizeparaffin-embedded samples and a glass-fiber filter to capture nucleicacids. QIAamp® DNA FFPE Tissue Kit uses QIAamp® DNA Micro technology forpurification of genomic and mitochondrial DNA.

The isolated nucleic acid samples (e.g., genomic DNA samples) can befragmented or sheared by practicing routine techniques. For example,genomic DNA can be fragmented by physical shearing methods, enzymaticcleavage methods, chemical cleavage methods, and other methods wellknown to those skilled in the art. The nucleic acid library can containall or substantially all of the complexity of the genome. The term“substantially all” in this context refers to the possibility that therecan in practice be some unwanted loss of genome complexity during theinitial steps of the procedure. The methods described herein also areuseful in cases where the nucleic acid library is a portion of thegenome, i.e., where the complexity of the genome is reduced by design.In some embodiments, any selected portion of the genome can be used withthe methods described herein. In certain embodiments, the entire exomeor a subset thereof is isolated.

Methods can further include isolating a nucleic acid sample to provide alibrary (e.g., a nucleic acid library). In certain embodiments, thenucleic acid sample includes whole genomic, subgenomic fragments, orboth. The isolated nucleic acid samples can be used to prepare nucleicacid libraries. Thus, in one embodiment, the methods featured in theinvention further include isolating a nucleic acid sample to provide alibrary (e.g., a nucleic acid library as described herein). Protocolsfor isolating and preparing libraries from whole genomic or subgenomicfragments are known in the art (e.g., 11lumina's genomic DNA samplepreparation kit). In certain embodiments, the genomic or subgenomic DNAfragment is isolated from a subject's sample (e.g., a tumor sample, anormal adjacent tissue (NAT), a blood sample or any normal control)). Inone embodiment, the sample (e.g., the tumor or NAT sample) is apreserved. For example, the sample is embedded in a matrix, e.g., anFFPE block or a frozen sample. In certain embodiments, the isolatingstep includes flow-sorting of individual chromosomes; and/ormicrodissecting a subject's sample (e.g., a tumor sample, a NAT, a bloodsample). In certain embodiments, the nucleic acid sample used togenerate the nucleic acid library is less than 5, less than 1 microgram,less than 500 ng, less than 200 ng, less than 100 ng, less than 50 ng orless than 20 ng (e.g., 10 ng or less).

In still other embodiments, the nucleic acid sample used to generate thelibrary includes RNA or cDNA derived from RNA. In some embodiments, theRNA includes total cellular RNA. In other embodiments, certain abundantRNA sequences (e.g., ribosomal RNAs) have been depleted. In someembodiments, the poly(A)-tailed mRNA fraction in the total RNApreparation has been enriched. In some embodiments, the cDNA is producedby random-primed cDNA synthesis methods. In other embodiments, the cDNAsynthesis is initiated at the poly(A) tail of mature mRNAs by priming byoligo(dT)-containing oligonucleotides. Methods for depletion, poly(A)enrichment, and cDNA synthesis are well known to those skilled in theart.

The method can further include amplifying the nucleic acid sample (e.g.,DNA or RNA sample) by specific or non-specific nucleic acidamplification methods that are well known to those skilled in the art.In some embodiments, certain embodiments, the nucleic acid sample isamplified, e.g., by whole-genome amplification methods such asrandom-primed strand-displacement amplification.

In other embodiments, the nucleic acid sample is fragmented or shearedby physical or enzymatic methods and ligated to synthetic adapters,size-selected (e.g., by preparative gel electrophoresis) and amplified(e.g., by PCR). In other embodiments, the fragmented and adapter-ligatedgroup of nucleic acids is used without explicit size selection oramplification prior to hybrid selection.

In other embodiments, the isolated DNA (e.g., the genomic DNA) isfragmented or sheared. In some embodiments, the library includes lessthan 50% of genomic DNA, such as a subfraction of genomic DNA that is areduced representation or a defined portion of a genome, e.g., that hasbeen subfractionated by other means. In other embodiments, the libraryincludes all or substantially all genomic DNA.

In some embodiments, the library includes less than 50% of genomic DNA,such as a subfraction of genomic DNA that is a reduced representation ora defined portion of a genome, e.g., that has been subfractionated byother means. In other embodiments, the library includes all orsubstantially all genomic DNA. Protocols for isolating and preparinglibraries from whole genomic or subgenomic fragments are known in theart (e.g., Illumina's genomic DNA sample preparation kit). AlternativeDNA shearing methods can be more automatable and/or more efficient(e.g., with degraded FFPE samples). Alternatives to DNA shearing methodscan also be used to avoid a ligation step during library preparation.

The methods described herein can be performed using a small amount ofnucleic acids, e.g., when the amount of source DNA is limiting (e.g.,even after whole-genome amplification). In one embodiment, the nucleicacid comprises less than about 5 μg, 4 μg, 3 μg, 2 μg, 1 μg, 0.8 μg, 0.7μg, 0.6 μg, 0.5 μg, or 400 ng, 300 ng, 200 ng, 100 ng, 50 ng, or 20 ngor less of nucleic acid sample. For example, to prepare 500 ng ofhybridization-ready nucleic acids, one typically begins with 3 μg ofgenomic DNA. One can start with less, however, if one amplifies thegenomic DNA (e.g., using PCR) before the step of solution hybridization.Thus it is possible, but not essential, to amplify the genomic DNAbefore solution hybridization.

In some embodiments, a library is generated using DNA (e.g., genomicDNA) from a sample tissue, and a corresponding library is generated withRNA (or cDNA) isolated from the same sample tissue.

Design of Baits

A bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule,which can hybridize to (e.g., be complementary to), and thereby allowcapture of a target nucleic acid. In one embodiment, a bait is an RNAmolecule. In other embodiments, a bait includes a binding entity, e.g.,an affinity tag, that allows capture and separation, e.g., by binding toa binding entity, of a hybrid formed by a bait and a nucleic acidhybridized to the bait. In one embodiment, a bait is suitable forsolution phase hybridization.

Baits can be produced and used by methods and hybridization conditionsas described in US 2010/0029498 and Gnirke, A. et al. (2009) NatBiotechnol. 27(2):182-189, and PCT/US 11/67725, filed Dec. 29, 2011,incorporated herein by reference. For example, biotinylated RNA baitscan be produced by obtaining a pool of synthetic long oligonucleotides,originally synthesized on a microarray, and amplifying theoligonucleotides to produce the bait sequences. In some embodiments, thebaits are produced by adding an RNA polymerase promoter sequence at oneend of the bait sequences, and synthesizing RNA sequences using RNApolymerase. In one embodiment, libraries of syntheticoligodeoxynucleotides can be obtained from commercial suppliers, such asAgilent Technologies, Inc., and amplified using known nucleic acidamplification methods.

Each bait sequence can include a target-specific (e.g., amember-specific) bait sequence and universal tails on each end. As usedherein, the term “bait sequence” can refer to the target-specific baitsequence or the entire oligonucleotide including the target-specific“bait sequence” and other nucleotides of the oligonucleotide. In oneembodiment, a target-specific bait hybridizes to a nucleic acid sequencecomprising a nucleic acid sequence in certain exons of a gene, e.g., acell cycle gene.

In one embodiment, the bait is an oligonucleotide about 200 nucleotidesin length, of which 170 nucleotides are target-specific “bait sequence.”The other 30 nucleotides (e.g., 15 nucleotides on each end) areuniversal arbitrary tails used for PCR amplification. The tails can beany sequence selected by the user. For example, the pool of syntheticoligonucleotides can include oligonucleotides of the sequence of5′-ATCGCACCAGCGTGTN₁₇₀CACTGCGGCTCCTCA-3′ (SEQ ID NO:1) with N₁₇₀indicating the target-specific bait sequences.

The bait sequences described herein can be used for selection of exonsand short target sequences. In one embodiment, the bait is between about100 nucleotides and 300 nucleotides in length. In another embodiment,the bait is between about 130 nucleotides and 230 nucleotides in length.In yet another embodiment, the bait is between about 150 nucleotides and200 nucleotides in length. The target-specific sequences in the baits,e.g., for selection of exons and short target sequences, are betweenabout 40 nucleotides and 1000 nucleotides in length. In one embodiment,the target-specific sequence is between about 70 nucleotides and 300nucleotides in length. In another embodiment, the target-specificsequence is between about 100 nucleotides and 200 nucleotides in length.In yet another embodiment, the target-specific sequence is between about120 nucleotides and 170 nucleotides in length.

Sequencing

The invention also includes methods of sequencing nucleic acids. In oneembodiment, any of a variety of sequencing reactions known in the artcan be used to directly sequence at least a portion of a mutant gene. Inone embodiment, the mutant gene sequence is compared to a correspondingreference (control) sequence.

In one embodiment, the sequence of the mutant nucleic acid molecule isdetermined by a method that includes one or more of: hybridizing anoligonucleotide, e.g., an allele specific oligonucleotide for onealteration described herein to said nucleic acid; hybridizing a primer,or a primer set (e.g., a primer pair), that amplifies a regioncomprising the mutation or a fusion junction of the allele; amplifying,e.g., specifically amplifying, a region comprising the mutation or afusion junction of the allele; attaching an adapter oligonucleotide toone end of a nucleic acid that comprises the mutation or a fusionjunction of the allele; generating an optical, e.g., a colorimetricsignal, specific to the presence of the one of the mutation or fusionjunction; hybridizing a nucleic acid comprising the mutation or fusionjunction to a second nucleic acid, e.g., a second nucleic acid attachedto a substrate; generating a signal, e.g., an electrical or fluorescentsignal, specific to the presence of the mutation or fusion junction; andincorporating a nucleotide into an oligonucleotide that is hybridized toa nucleic acid that contains the mutation or fusion junction.

In another embodiment, the sequence is determined by a method thatcomprises one or more of: determining the nucleotide sequence from anindividual nucleic acid molecule, e.g., where a signal corresponding tothe sequence is derived from a single molecule as opposed, e.g., from asum of signals from a plurality of clonally expanded molecules;determining the nucleotide sequence of clonally expanded proxies forindividual nucleic acid molecules; massively parallel short-readsequencing; template-based sequencing; pyrosequencing; real-timesequencing comprising imaging the continuous incorporation ofdye-labeling nucleotides during DNA synthesis; nanopore sequencing;sequencing by hybridization; nano-transistor array based sequencing;polony sequencing; scanning tunneling microscopy (STM) based sequencing;or nanowire-molecule sensor based sequencing.

Any method of sequencing known in the art can be used. Exemplarysequencing reactions include those based on techniques developed byMaxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger(Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety ofautomated sequencing procedures can be utilized when performing theassays (Biotechniques (1995) 19:448), including sequencing by massspectrometry (see, for example, U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/16101,entitled DNA Sequencing by Mass Spectrometry by H. Koster; U.S. Pat. No.5,547,835 and international patent application Publication Number WO94/21822 entitled DNA Sequencing by Mass Spectrometry Via ExonucleaseDegradation by H. Koster), and U.S. Pat. No. 5,605,798 and InternationalPatent Application No. PCT/US96/03651 entitled DNA Diagnostics Based onMass Spectrometry by H. Koster; Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159).

Sequencing of nucleic acid molecules can also be carried out usingnext-generation sequencing (NGS). Next-generation sequencing includesany sequencing method that determines the nucleotide sequence of eitherindividual nucleic acid molecules or clonally expanded proxies forindividual nucleic acid molecules in a highly parallel fashion (e.g.,greater than 10⁵ molecules are sequenced simultaneously). In oneembodiment, the relative abundance of the nucleic acid species in thelibrary can be estimated by counting the relative number of occurrencesof their cognate sequences in the data generated by the sequencingexperiment. Next generation sequencing methods are known in the art, andare described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews11:31-46, incorporated herein by reference.

In one embodiment, the next-generation sequencing allows for thedetermination of the nucleotide sequence of an individual nucleic acidmolecule (e.g., Helicos BioSciences' HeliScope Gene Sequencing system,and Pacific Biosciences' PacBio RS system). In other embodiments, thesequencing method determines the nucleotide sequence of clonallyexpanded proxies for individual nucleic acid molecules (e.g., the Solexasequencer, Illumina Inc., San Diego, Calif.; 454 Life Sciences(Branford, Conn.), and Ion Torrent). e.g., massively parallel short-readsequencing (e.g., the Solexa sequencer, Illumina Inc., San Diego,Calif.), which generates more bases of sequence per sequencing unit thanother sequencing methods that generate fewer but longer reads. Othermethods or machines for next-generation sequencing include, but are notlimited to, the sequencers provided by 454 Life Sciences (Branford,Conn.), Applied Biosystems (Foster City, Calif.; SOLiD sequencer), andHelicos BioSciences Corporation (Cambridge, Mass.).

Platforms for next-generation sequencing include, but are not limitedto, Roche/454's Genome Sequencer (GS) FLX System, Illumina/Solexa'sGenome Analyzer (GA), Life/APG's Support Oligonucleotide LigationDetection (SOLiD) system, Polonator's G.007 system, Helicos BioSciences'HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RSsystem.

NGS technologies can include one or more of steps, e.g., templatepreparation, sequencing and imaging, and data analysis.

Template Preparation

Methods for template preparation can include steps such as randomlybreaking nucleic acids (e.g., genomic DNA or cDNA) into smaller sizesand generating sequencing templates (e.g., fragment templates ormate-pair templates). The spatially separated templates can be attachedor immobilized to a solid surface or support, allowing massive amountsof sequencing reactions to be performed simultaneously. Types oftemplates that can be used for NGS reactions include, e.g., clonallyamplified templates originating from single DNA molecules, and singleDNA molecule templates.

Methods for preparing clonally amplified templates include, e.g.,emulsion PCR (emPCR) and solid-phase amplification.

EmPCR can be used to prepare templates for NGS. Typically, a library ofnucleic acid fragments is generated, and adapters containing universalpriming sites are ligated to the ends of the fragment. The fragments arethen denatured into single strands and captured by beads. Each beadcaptures a single nucleic acid molecule. After amplification andenrichment of emPCR beads, a large amount of templates can be attachedor immobilized in a polyacrylamide gel on a standard microscope slide(e.g., Polonator), chemically crosslinked to an amino-coated glasssurface (e.g., Life/APG; Polonator), or deposited into individualPicoTiterPlate (PTP) wells (e.g., Roche/454), in which the NGS reactioncan be performed.

Solid-phase amplification can also be used to produce templates for NGS.Typically, forward and reverse primers are covalently attached to asolid support. The surface density of the amplified fragments is definedby the ratio of the primers to the templates on the support. Solid-phaseamplification can produce hundreds of millions spatially separatedtemplate clusters (e.g., Illumina/Solexa). The ends of the templateclusters can be hybridized to universal sequencing primers for NGSreactions.

Other methods for preparing clonally amplified templates also include,e.g., Multiple Displacement Amplification (MDA) (Lasken R. S. Curr OpinMicrobiol. 2007; 10(5):510-6). MDA is a non-PCR based DNA amplificationtechnique. The reaction involves annealing random hexamer primers to thetemplate and DNA synthesis by high fidelity enzyme, typically 029 at aconstant temperature. MDA can generate large sized products with lowererror frequency.

Template amplification methods such as PCR can be coupled with NGSplatforms to target or enrich specific regions of the genome (e.g.,exons). Exemplary template enrichment methods include, e.g.,microdroplet PCR technology (Tewhey R. et al., Nature Biotech. 2009,27:1025-1031), custom-designed oligonucleotide microarrays (e.g.,Roche/NimbleGen oligonucleotide microarrays), and solution-basedhybridization methods (e.g., molecular inversion probes (MIPs) (PorrecaG. J. et al., Nature Methods, 2007, 4:931-936; Krishnakumar S. et al.,Proc. Natl. Acad. Sci. USA, 2008, 105:9296-9310; Turner E. H. et al.,Nature Methods, 2009, 6:315-316), and biotinylated RNA capture sequences(Gnirke A. et al., Nat. Biotechnol. 2009; 27(2):182-9)

Single-molecule templates are another type of templates that can be usedfor NGS reaction. Spatially separated single molecule templates can beimmobilized on solid supports by various methods. In one approach,individual primer molecules are covalently attached to the solidsupport. Adapters are added to the templates and templates are thenhybridized to the immobilized primers. In another approach,single-molecule templates are covalently attached to the solid supportby priming and extending single-stranded, single-molecule templates fromimmobilized primers. Universal primers are then hybridized to thetemplates. In yet another approach, single polymerase molecules areattached to the solid support, to which primed templates are bound.

Sequencing and Imaging

Exemplary sequencing and imaging methods for NGS include, but are notlimited to, cyclic reversible termination (CRT), sequencing by ligation(SBL), single-molecule addition (pyrosequencing), and real-timesequencing.

CRT uses reversible terminators in a cyclic method that minimallyincludes the steps of nucleotide incorporation, fluorescence imaging,and cleavage. Typically, a DNA polymerase incorporates a singlefluorescently modified nucleotide corresponding to the complementarynucleotide of the template base to the primer. DNA synthesis isterminated after the addition of a single nucleotide and theunincorporated nucleotides are washed away. Imaging is performed todetermine the identity of the incorporated labeled nucleotide. Then inthe cleavage step, the terminating/inhibiting group and the fluorescentdye are removed. Exemplary NGS platforms using the CRT method include,but are not limited to, Illumina/Solexa Genome Analyzer (GA), which usesthe clonally amplified template method coupled with the four-color CRTmethod detected by total internal reflection fluorescence (TIRF); andHelicos BioSciences/HeliScope, which uses the single-molecule templatemethod coupled with the one-color CRT method detected by TIRF.

SBL uses DNA ligase and either one-base-encoded probes ortwo-base-encoded probes for sequencing. Typically, a fluorescentlylabeled probe is hybridized to its complementary sequence adjacent tothe primed template. DNA ligase is used to ligate the dye-labeled probeto the primer. Fluorescence imaging is performed to determine theidentity of the ligated probe after non-ligated probes are washed away.The fluorescent dye can be removed by using cleavable probes toregenerate a 5′-PO₄ group for subsequent ligation cycles. Alternatively,a new primer can be hybridized to the template after the old primer isremoved. Exemplary SBL platforms include, but are not limited to,Life/APG/SOLiD (support oligonucleotide ligation detection), which usestwo-base-encoded probes.

Pyrosequencing method is based on detecting the activity of DNApolymerase with another chemiluminescent enzyme. Typically, the methodallows sequencing of a single strand of DNA by synthesizing thecomplementary strand along it, one base pair at a time, and detectingwhich base was actually added at each step. The template DNA isimmobile, and solutions of A, C, G, and T nucleotides are sequentiallyadded and removed from the reaction. Light is produced only when thenucleotide solution complements the first unpaired base of the template.The sequence of solutions which produce chemiluminescent signals allowsthe determination of the sequence of the template. Exemplarypyrosequencing platforms include, but are not limited to, Roche/454,which uses DNA templates prepared by emPCR with 1-2 million beadsdeposited into PTP wells.

Real-time sequencing involves imaging the continuous incorporation ofdye-labeled nucleotides during DNA synthesis. Exemplary real-timesequencing platforms include, but are not limited to, PacificBiosciences platform, which uses DNA polymerase molecules attached tothe surface of individual zero-mode waveguide (ZMW) detectors to obtainsequence information when phospholinked nucleotides are beingincorporated into the growing primer strand; Life/VisiGen platform,which uses an engineered DNA polymerase with an attached fluorescent dyeto generate an enhanced signal after nucleotide incorporation byfluorescence resonance energy transfer (FRET); and LI-COR Biosciencesplatform, which uses dye-quencher nucleotides in the sequencingreaction.

Other sequencing methods for NGS include, but are not limited to,nanopore sequencing, sequencing by hybridization, nano-transistor arraybased sequencing, polony sequencing, scanning tunneling microscopy (STM)based sequencing, and nanowire-molecule sensor based sequencing.

Nanopore sequencing involves electrophoresis of nucleic acid moleculesin solution through a nano-scale pore which provides a highly confinedspace within which single-nucleic acid polymers can be analyzed.Exemplary methods of nanopore sequencing are described, e.g., in BrantonD. et al., Nat Biotechnol. 2008; 26(10):1146-53.

Sequencing by hybridization is a non-enzymatic method that uses a DNAmicroarray. Typically, a single pool of DNA is fluorescently labeled andhybridized to an array containing known sequences. Hybridization signalsfrom a given spot on the array can identify the DNA sequence. Thebinding of one strand of DNA to its complementary strand in the DNAdouble-helix is sensitive to even single-base mismatches when the hybridregion is short or is specialized mismatch detection proteins arepresent. Exemplary methods of sequencing by hybridization are described,e.g., in Hanna G. J. et al., J. Clin. Microbiol. 2000; 38 (7): 2715-21;and Edwards J. R. et al., Mut. Res. 2005; 573 (1-2): 3-12.

Polony sequencing is based on polony amplification andsequencing-by-synthesis via multiple single-base-extensions (FISSEQ).Polony amplification is a method to amplify DNA in situ on apolyacrylamide film. Exemplary polony sequencing methods are described,e.g., in US Patent Application Publication No. 2007/0087362.

Nano-transistor array based devices, such as Carbon NanoTube FieldEffect Transistor (CNTFET), can also be used for NGS. For example, DNAmolecules are stretched and driven over nanotubes by micro-fabricatedelectrodes. DNA molecules sequentially come into contact with the carbonnanotube surface, and the difference in current flow from each base isproduced due to charge transfer between the DNA molecule and thenanotubes. DNA is sequenced by recording these differences. ExemplaryNano-transistor array based sequencing methods are described, e.g., inU.S. Patent Application Publication No. 2006/0246497.

Scanning tunneling microscopy (STM) can also be used for NGS. STM uses apiezo-electric-controlled probe that performs a raster scan of aspecimen to form images of its surface. STM can be used to image thephysical properties of single DNA molecules, e.g., generating coherentelectron tunneling imaging and spectroscopy by integrating scanningtunneling microscope with an actuator-driven flexible gap. Exemplarysequencing methods using STM are described, e.g., in U.S. PatentApplication Publication No. 2007/0194225.

A molecular-analysis device which is comprised of a nanowire-moleculesensor can also be used for NGS. Such device can detect the interactionsof the nitrogenous material disposed on the nanowires and nucleic acidmolecules such as DNA. A molecule guide is configured for guiding amolecule near the molecule sensor, allowing an interaction andsubsequent detection. Exemplary sequencing methods usingnanowire-molecule sensor are described, e.g., in U.S. Patent ApplicationPublication No. 2006/0275779.

Double ended sequencing methods can be used for NGS. Double endedsequencing uses blocked and unblocked primers to sequence both the senseand antisense strands of DNA. Typically, these methods include the stepsof annealing an unblocked primer to a first strand of nucleic acid;annealing a second blocked primer to a second strand of nucleic acid;elongating the nucleic acid along the first strand with a polymerase;terminating the first sequencing primer; deblocking the second primer;and elongating the nucleic acid along the second strand. Exemplarydouble ended sequencing methods are described, e.g., in U.S. Pat. No.7,244,567.

Data Analysis

After NGS reads have been generated, they can be aligned to a knownreference sequence or assembled de novo.

For example, identifying genetic variations such as single-nucleotidepolymorphism and structural variants in a sample (e.g., a tumor sample)can be accomplished by aligning NGS reads to a reference sequence (e.g.,a wild-type sequence). Methods of sequence alignment for NGS aredescribed e.g., in Trapnell C. and Salzberg S. L. Nature Biotech., 2009,27:455-457.

Examples of de novo assemblies are described, e.g., in Warren R. et al.,Bioinformatics, 2007, 23:500-501; Butler J. et al., Genome Res., 2008,18:810-820; and Zerbino D. R. and Birney E., Genome Res., 2008,18:821-829.

Sequence alignment or assembly can be performed using read data from oneor more NGS platforms, e.g., mixing Roche/454 and Illumina/Solexa readdata.

Algorithms and methods for data analysis are described in U.S. Ser. No.61/428,568, filed Dec. 30, 2010, incorporated herein by reference.

Screening Methods

In another aspect, the invention features a method, or assay, forscreening for agents that modulate, e.g., inhibit, the expression oractivity of a mutant gene, e.g., a mutant cell cycle gene, such asCDKN2A, CDKN2B or CCND1 as described herein. The method includescontacting a mutant polypeptide, or a cell expressing a mutant gene,with a candidate agent; and detecting a change in a parameter associatedwith the mutant gene, e.g., a change in the expression or an activity ofthe mutant gene. The method can, optionally, include comparing thetreated parameter to a reference value, e.g., a control sample (e.g.,comparing a parameter obtained from a sample with the candidate agent toa parameter obtained from a sample without the candidate agent). In oneembodiment, if a decrease in expression or activity of the mutant geneis detected, the candidate agent is identified as an inhibitor. Inanother embodiment, if an increase in expression or activity of themutant gene is detected, the candidate agent is identified as anactivator. In certain embodiments, the mutant gene is a nucleic acidmolecule or a polypeptide as described herein.

In one embodiment, the contacting step is effected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is effected in a cell in culture, e.g.,a cell expressing a mutant gene, such as a mutant cell cycle gene (e.g.,a mammalian cell, a tumor cell or cell line, a recombinant cell). In yetother embodiments, the contacting step is effected in a cell in vivo (amutant gene-expressing cell present in a subject, e.g., an animalsubject (e.g., an in vivo animal model).

Exemplary parameters evaluated include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to a mutant polypeptide; a binding competition between a knownligand and the candidate agent to a mutant polypeptide;

(ii) a change in transcriptional activation activity or DNA bindingactivity as measured, for example, by fusing a response element to areporter gene; DNA binding activity can also be measure by gel-shiftassay;

(iii) a change in an activity of a cell containing a mutant gene (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology or tumorigenicity of the cell;

(iv) a change in tumor present in an animal subject, e.g., size,appearance, proliferation, of the tumor; or

(v) a change in the level, e.g., expression level, of a mutantpolypeptide or nucleic acid molecule.

EXAMPLES Example 1: Massively Parallel Sequencing Assays to IdentifyNovel Alterations

The following exemplifies the use of massively parallel sequencingassays to identify mutations in biopsy samples from patients with HNSCC(Head and Neck Squamous Cell Carcinoma).

Massively parallel sequencing technology was used to examine formalinfixed paraffin embedded (FFPE) samples of HNSCC. This assay can identifyall classes of DNA alterations (e.g., base substitutions, insertions anddeletions, copy number alterations and rearrangements) in a singlediagnostic test. Patient samples were analyzed according to whether thepatient was positive for human papillomavirus (HPV⁺) or negative for HPV(HPV⁻).

FIG. 1 is a summary of the result of the sequencing analysis. Most ofthe mutant genes identified carried either point mutations or mutationsthat resulted in copy number alterations. Samples contained pointmutations in certain genes or mutations that resulted in copy numberalterations (CNA). In some HPV+ patients, both types of mutations wereidentified in the PIK3CA gene.

The summary of mutations observed is shown in Tables 1 and 4.

TABLE 1 Summary of mutations observed in HPV+ and HPV− patients HPV+HPV− % samples % samples carrying carrying Gene Mutation type mutationMutation Type mutation PIK3CA^(a) Point mutations ~33% Point mutations~22% and/or CNA or CNA FBXW7 Point mutations ~19% CNA ~5% PTEN^(a) Pointmutations ~19% Point mutations ~6% or CNA SOX2 CNA ~16% CNA ~6% BCL2L1CNA ~12% — 0% KRAS Point mutations ~12% — 0% NF1 Point mutations ~8% —0% RB1 Point mutations ~4% — 0% STK11^(a) Point mutations ~4% — 0%NOTCH1 Point mutations ~4% Point mutations ~5% RICTOR CNA ~4% CNA ~5%FGFR1 — 0% CNA ~5% MCL1 — 0% CNA ~5% MYC — 0% CNA ~5% FGFR3 — 0% Pointmutations ~5% KDM6A — 0% Point mutations ~5% LRP1B — 0% Point mutations~5% PKHD1 — 0% Point mutations ~5% SUFU — 0% Point mutations ~5% TET2 —0% Point mutations ~5% MDM2 — 0% CNA ~12% EGFR — 0% CNA ~16% CDKN2A^(b)— 0% Point mutations ~50% or CNA CCND1^(b) CNA ~4% CNA ~65% TP53 Pointmutations ~6% Point mutations ~100% ^(a)PI3K pathway gene ^(b)cell cyclegene

Materials and Methods

Sample Collection, p16 Staining and DNA Extraction.

Twenty HPV+ and twenty HPV− oropharyngeal carcinomas were selected, allformalin fixed paraffin-embedded (Table 2). HPV status was confirmed byp16 staining, and by quantitative PCR for HPV−16 E6, having been shownto have 97% sensitivity, 94% specificity and to be the bestdiscriminator of favourable outcome. Sequencing demonstrated 100%concordance of HPV status. All samples were laser-capturedmicrodissected (LCM) to separate tumor epithelial from surroundingstromal tissues, enriching tumor DNA for further analyses. These wereprocessed as 10 μm thick unstained slides which were reviewed by anexpert pathologist who had marked the slides for tumor subtypeenrichment in a corresponding H&E stained section. LCM was carried outon P.A.L.M. MembraneSlide 1.0 PEN slides (Zeiss Microimaging, Munich,Germany) using the Zeiss Palm Microbeam™ system. Tissue was collectedinto extraction tubes and processed using the QIAamp DNA FFPE Tissue Kit(Qiagen, Hilden, Germany). Extracted DNA was quantified using astandardized PicoGreen fluorescence assay (LifeTechnologies, Carlsbad,Calif.).

TABLE 2 Demographics of twenty HPV+ and twenty HPV− oropharyngealcarcinomas selected for analysis. HPV+ (n = 20) HPV− (n = 20) Median Age56.5 years (42-81) 58 years (45-77) (range) Tumor Site Oropharynx: 20Oropharynx: 20 Tumor Grade Well diff: 1 Well diff: 0 Mod diff: 9 Moddiff: 16 Poorly diff: 10 Poorly diff: 4 Tumor T1; 5 T1; 1 Stage (T) T2:8 T2: 4 T3: 3 T3: 5 T4: 3 T4: 10 N/a: 1 N/a: 0 Cervical Yes: 16 Yes: 13Lymph Node No: 2 No: 6 Involvement N/a: 2 N/a: 1 Smoking Ever: 9 Ever:15 Never: 8 Never: 0 N/a: 3 N/a: 5 Alcohol Heavy Drinker Heavy Drinker(>20 U/w): 2 (>20 U/w): 12 Occ alcohol: 5 Occ alcohol: 3 Never: 4 Never:0 N/a: 9 N/a: 5

DNA Library Construction and Hybrid Capture.

Molecular barcode-indexed ligation-based sequencing libraries wereconstructed using 50-200 ng of total genomic DNA recovered from thesample. Libraries were hybridization captured with custom biotinylatedRNA oligo pools (custom SureSelect kit, Agilent) representing 3,230exons in 182 cancer-related genes plus 37 introns from 14 genes oftenrearranged in cancer (FIGS. 2A and 2B).

Sequencing and Analysis.

Paired end sequencing (49×49 cycles) was performed using the HiSeq2000(Illumina). Sequence data from gDNA, available from 18 HPV+ and 16 HPV−samples, were mapped to the reference human genome (hg19) using the BWAaligner (Li et al. Bioinformatics 25:2078-9) and processed usingpublicly available SAMtools (Li et al., Bioinformatics 25:2078-9, 2009),Picard (online at picard.sourceforge.net) and GATK (McKenna et al.,Genome Res. 20-1297-303, 2010). Genomic base substitutions and indelswere detected using custom tools optimized for mutation calling inheterogeneous tumor samples, based on statistical modeling of sequencequality scores and local sequence assembly. Variations were filteredusing dbSNP_135_ENREF_1 (online at ncbi.nlm.nih.gov/projects/SNP/) and acustom artifact database, then annotated for known and likely somaticmutations using the COSMIC (Forbes et al., Nucl. Acids Res. 39:D945-50,2011). Copy number alterations were detected by comparing targetedgenomic DNA sequence coverage with a process-matched normal controlsample. Genomic rearrangements were detected by clustering chimericreads mapping to targeted introns.

Validation of Selected Mutations by Sequenom OncoCarta.

DNA extracted from FFPE samples were sent to Sequenom (Hamburg, Germany)for blind testing and analysis, using Sequenom OncoCarta panels v1.0 andv3.0, as previously described (Fumagalli et al., BMC Cancer 10:101,2010).

Confirmation of Copy Number Changes by Infinium CNV Profiling.

Using previously obtained Infinium HumanMethylation450 BeadChipmethylation data on sequenced samples (manuscript submitted), theBioconductor package ‘DNAcopy’ (online atbioc.ism.ac.jp/2.10/bioc/html/DNAcopy.html) was applied to calculate thecopy number of the majority of sequenced samples.

Immunohistochemistry and Interpretation of Results.

All sequenced HNSCC samples were stained for PTEN and for Cyclin D1.Antibody 04-409 (Millipore-Merck KGaA, Darmstadt, Germany) was used forPTEN staining and antibody P2D11F11 (Novocastra) was used for Cyclin D1staining of 10 μm thick slides. The stained slides were examined andscored as previously described (Djordjevic et al., Mod. Pathol.25:699-708, 2012), by two experienced histopathologists.

Results

Patient Demographic Data.

The median age was slightly higher in the HPV− group (58 vs. 56.5 years)(Table 2). Male to female ratio was similar between the Groups, and themajority of cases show moderately or poorly differentiated histologywith evidence of lymph nodal involvement at presentation. The vastmajority of HPV− cases were in active smokers and/or heavy alcohol users(FIGS. 7A and 7B).

Next Generation Sequencing.

Sequence analysis revealed that HPV+ and HPV− oropharyngeal carcinomascluster into two distinct subgroups, with few overlapping geneticalterations (FIG. 3A). TP53 mutations were detected in 100% of HPV−samples. The list of observed TP53 mutations is illustrated in Table 3.Samples did not cluster into two distinct subgroups after exclusion ofthe TP53 mutation data (FIG. 3B). Copy number alterations in CCND1amplification and CDKN2A/B deletions were exclusively detected in HPV−cases (in around 55% and 40% of cases respectively). PIK3CA mutation oramplification, and PTEN inactivation by gene copy loss or mutation wereseen in over 60% of HPV+ tumors, and in 31% HPV− tumors. FBXW7alterations were present in over 15% of all samples, and SOX2amplifications were observed in 12% of cases.

TABLE 3 TP53 Mutations Observed TP53 Sample Name Mutations P6_pos R290CP8_pos None P13_pos None P19_pos None P26_pos None P28_pos None P35_posNone P38_pos None P43_pos None P50_pos None P60_pos None P67_pos NoneP72_pos None P74_pos None P79_pos None P82_pos None P83_pos NoneP105_pos None P7_pos R175H P10_pos Y234H P12_pos Y220S P14_pos R273LP17_pos G154fs P24_pos L130fs P25_pos Q165 P29_pos Y236 P40_pos R306P62_pos Y220C P70_pos Q104 P90_pos L114fs, L330fs P91_pos R337L P92_posR335L, G334V P94_pos T155P P95_pos 920-1C > T splice

Validation of Obtained Results.

Copy number gains and losses detected by NGS were interrogated byInfinium CNV profiling. 42 of 44 (95%) copy number alterations detectedby sequencing were confirmed (FIG. 4). In order to obtain a globalpicture, copy number alterations were detected by Infinium CNVprofiling. Comparing the obtained genome-wide copy number alterationprofiles between HPV+ and HPV− cancers revealed that overall, similargenomic regions harbor concordant copy number changes in both groups (inparticular in chromosomes 3, 7 and 14). Amplification of 3q seemed to bea particular target in both HPV+ and HPV− HNSCC lesions (FIG. 5).

The mutations detected by NGS were validated by Sequenom OncoCartapanels v1.0 and v3.0. As the NGS exome sequencing targeted the wholegene sequence, and the Sequenom OncoCarta panels targeted only specificmutational hotspots of certain genes, the majority of NGS detectedmutations were not included in the Sequenom analysis. Eight out of 9mutations that were detected by NGS were also confirmed by Sequenom. OnePIK3CA mutation in sample P72_pos was called at 1% allele frequency byNGS, and this mutation was therefore unlikely to be detected by Sequenomanalysis.

For CCND1 and PTEN the findings were also validated byimmunohistochemistry. Genomic alterations in CCND1 were confirmed byCyclin D1 immunochemistry with strong expression of Cyclin D1 protein in8 of 9 CCND1 amplified cases (and intermediate expression in theremaining case). PTEN loss and mutation was validated byimmunohistochemistry. PTEN staining was negative in all cases in whichdeep sequencing revealed a homozygous deletion or mutation. Fouradditional samples displayed low PTEN protein expression. In three ofthese cases a heterozygous deletion/single copy loss of PTEN waspresent, as detected by NGS. In the remaining sample other mechanismsmay explain the loss of expression, such as an epigenetic alteration orchanges in the posttranscriptional regulation of PTEN.

Integration with Mutation Data from Lung Cancer.

Genetic changes described in lung adenocarcinoma in previous studies(Ding et al., Nature 455:1069-75, 2008) were compared to our data fromHPV− HNSCC (FIG. 6).

We discovered that HPV⁻ HNSCC patients are more likely to carrymutations in cell cycle genes, such as CDKN2A or CDKN2B, or CCND1, thanare HPV⁺ patients, and that HPV⁺ HNSCC patients are more likely to carrymutations in genes in the phosphatidylinositol-3 kinase (PI3K) family,such as PIK3CA, PTEN and STK11. Thus, an HNSCC patient diagnosed as HPV⁻can be administered cell cycle inhibitors, such as cdk (cyclin-dependentkinase) inhibitors.

Overall, sequence analysis revealed that HPV+ and HPV− oropharyngealcarcinomas cluster into two distinct subgroups, with few overlappinggenetic alterations. These data concur with epidemiological and clinicaldata, indicating that HPV+ HNSCC is a distinct disease entity.

The fact that targeted deep next generation sequencing revealed thatTP53 is mutated in 100% of HPV− samples indicates that TP53 is likely tobe a ubiquitous early event in the pathogenesis of oropharyngeal HNSCCcaused by smoking, and/or alcohol use. In HPV+ disease, E6 leads to TP53functional inactivation. Consistent with this, only one TP53 mutationwas identified in an HPV+ tumor. Furthermore, this mutation (R290C,Table 3) caused only a 40% decrease in TP53 function.

The data for HPV− oropharyngeal cancer indicated that the frequency ofCCND1 amplifications (in around 55% of cases) and CDKN2A/B deletions (inaround 40% of cases) were higher than previously reported. In HPV+cancer, the oncoprotein E7 leads to cell cycle dysregulation bysubstituting for cyclin D gain-of-function and cyclin dependent kinaseinhibitor loss-of-function activities. Overall, this indicates thatdirect dysregulation of the cell cycle is a key mechanism fororopharyngeal tumors to evolve.

We demonstrated for the first time inactivating mutations in NF1 andSTK11 in HPV+ HNSCC.

Overall, our data strongly support a causal role for HPV inoropharyngeal carcinogenesis by overcoming the requirement for geneticlesions in the TP53 and RB1 tumor suppressor pathways evident in theHPV− tumors.

TABLE 4 Column 3 somatic mutations¹ Amplification of (mutant allelegenes known to be Deletions of genes HPV frequency/ Column 4 somaticamplified in cancer known to be deleted Tissue type status coveragedepth) mutations² (CN level, exon span) in cancer Oropharynx posFBXW7_c.1436G > none PIK3CA_gain (6, PIK3CA_target_6- none (base ofA_p.R479Q (0.10, 1868), 20); tongue) FBXW7_c.1513C > SOX2_gain (6,SOX2_target_1- T_p.R505C (0.12, 2417), 1) FBXW7_c.1514G > A_p.R505H(0.08, 2429), PIK3CA_c.1624G > A_p.E542K (0.26, 3391), STK11_c.971C >T_p. P324L (0.03, 711), TP53_c.868C > T_p. R290C (0.06, 762) — —FBXW7_c.1436G > none PIK3CA_gain (5, PIK3CA_target_6- none A_p.R479Q(0.10, 535), 20); FBXW7_c.1513C > SOX2_gain (5, SOX2_target_1- T_p.R505C(0.13, 625), 1) FBXW7_c.1514G > A_p.R505H (0.08, 625), PIK3CA_c.1624G >A_p.E542K (0.27, 990), TP53_c.868C > T_p. R290C (0.10, 225) Oropharynxneg PIK3CA_c.1035T > none none CDKN2A_loss (0, (tonsil) A_p.N345K (0.38,1356), CDKN2A_target_3-4); TP53_c.524G > A_p. CDKN2B_loss (0, R175H(0.27, 535) CDKN2B_target_1-2) Oropharynx pos FBXW7_c.1436G > none nonenone (tonsil) A_p.R479Q (0.41, 630) Oropharynx neg TP53_c.700T > C_p.none none none (tonsil) Y234H (0.16, 221) Oropharynx neg PKHD1_c.3241C >T_p. none CCND1_gain (7, CCND1_target_1- none (base of R1081C (0.62,406), 5); tongue) TP53_c.659A > C_p. MDM2_gain (7, MDM2_target_1- Y220S(0.84, 420) 11) Oropharynx pos FBXW7_c.1513C > none none PTEN_loss (0,(tonsil) T_p.R505C (0.35, 402) PTEN_target_1-9) Oropharynx negTP53_c.818G > T_p. none CCND1_gain (12, CCND1_target_1- none (tonsil)R273L (0.65, 390) 5); CCND3_gain (14, CCND3_target_1- 5); MCL1_gain (12,MCL1_target_1- 3); PIK3CA_gain (7, PIK3CA_target_1- 20); SOX2_gain (7,SOX2_target_1- 1) Oropharynx neg PIK3CA_c.1633G > TET2_c.4333C > T_p.CCND1_gain (13, CCND1_target_1- CDKN2A_loss (0, (tonsil) A_p.E545K(0.28, 1699), Q1445* (0.04, 984) 5) CDKN2A_target_1-4);TP53_c.456delG_p. CDKN2B_loss (0, G154fs*16 (0.65, CDKN2B_target_1-2)594) Oropharynx pos PIK3CA_c.1624G > none none none (tonsil) A_p.E542K(0.07, 933) Oropharynx neg none TP53:NM_000546: none CDKN2A_loss (0,(tonsil) c.388_392delTTGAG_p. CDKN2A_target_1-4); L130fs*17:frameshiftCDKN2B_loss (0, (0.70, 183) CDKN2B_target_1-2) Oropharynx negTP53_c.493C > T_p. none CCND1_gain (16, CCND1_target_1- CDKN2A_loss (0,(base of Q165* (0.69, 617) 5); CDKN2A_target_1-4); tongue) MYC_gain (8,MYC_target_1- CDKN2B_loss (0, 3) CDKN2B_target_1-2) Oropharynx pos nonenone none none (tonsil/base of tongue) Oropharynx pos none none nonenone (tonsil/base of tongue) Oropharynx neg FGFR3_c.746C > G_p. noneFGFR1_gain (10, FGFR1_target_1- KDM6A_loss (0, (tonsil/base S249C (0.15,550), 17) KDM6A_target_1-29) of tongue) TP53_c.708C > A_p. Y236* (0.64,294) Oropharynx pos NF1_c.3721C > T_p. none none PTEN_loss (0, (tonsil)R1241* (0.02, 1429), PTEN_target_1-9) NF1_c.4600C > T_p. R1534* (0.01,1911) Oropharynx pos none none none (tonsil) Oropharynx negTP53_c.916C > T_p. PTEN:NM_000314: CCND1_gain (8, CCND1_target_1-CDKN2A_loss (0, (base of R306* (0.84, 710) c.79 + 1G > T:splice 5)CDKN2A_target_1-4); tongue) (0.81, 782) CDKN2B_loss (0,CDKN2B_target_1-2); FBXW7_loss (0, FBXW7_target_1-13) Oropharynx posnone none none none (tonsil) Oropharynx pos KRAS_c.35G > A_p. none nonenone (tonsil/base G12D (0.25, 513) of tongue) Oropharynx pos none nonenone PTEN_loss (0, (tonsil) PTEN_target_1-9) Oropharynx negTP53_c.659A > G_p. CDKN2A:NM_000077: CCND1_gain (15, CCND1_target_1-none (tonsil) Y220C (0.55, 112) c.458 − 5) 1C > T:splice (0.46, 91),PIK3CA_c.3085G > C_p.D1029H (0.18, 184) Oropharynx pos none noneBCL2L1_gain (11, BCL2L1_target_1- none (base of 1); tongue) PIK3CA_gain(8, PIK3CA_target_6- 20); SOX2_gain (8, SOX2_target_1- 1) Oropharynx negTP53_c.310C > T_p. LRP1B:NM_018557: none none (tonsil) Q104* (0.67, 479)c.7154_7155insT_p. I2387fs*2:frameshift (0.17, 364), SUFU:NM_016169:c.30_30delC_p. G11fs*85:frameshift (0.34, 822) Oropharynx posPIK3CA_c.1633G > none PIK3CA_gain (6, PIK3CA_target_1- none (tonsil)A_p.E545K (0.01, 1853) 20); SOX2_gain (6, SOX2_target_1- 1) Oropharynxpos none none BCL2L1_gain (11, BCL2L1_target_1- none (base of 1) tongue)Oropharynx pos PTEN_c.733C > T_p. none none none (tonsil) Q245* (0.38,393) Oropharynx pos PIK3CA_c.3140A > none none none (tonsil) T_p.H1047L(0.24, 695) Oropharynx pos NF1_c.3721C > T_p. NF1:NM_001042492: nonenone (tonsil) R1241* (0.06, 493), c.4700C > A_p.S1567* RB1_c.1735C >T_p. (0.06, 580), R579* (0.41, 288) PIK3CA_c.2176G > A_p.E726K (0.49,420) Oropharynx neg none TP53:NM_000546: EGFR_gain (7, EGFR_target_1-CDKN2A_loss (0, (tonsil) c.339_340insA_p. 29) CDKN2A_target_1-4);L114fs*35:frameshift CDKN2B_loss (0, (0.60, 306) CDKN2B_target_1-2)Oropharynx neg TP53_c.1010G > T_p. TP53:NM_000546: CCND1_gain (16,CCND1_target_1- (base of R337L (0.29, 127) c.986_986delG_p. 5) tongue)L330fs*15:frameshift (0.18, 197) Oropharynx neg TP53_c.1001G > T_p. noneCCND1_gain (7, CCND1_target_1- none (base of G334V (0.73, 95), 5)tongue) TP53_c.1004G > T_p. R335L (0.74, 92) Oropharynx negTP53_c.463A > C_p. none RICTOR_gain (7, RICTOR_target_1- none (base ofT155P (0.62, 121) 39) tongue) Oropharynx neg none TP53:NM_000546:CCND1_gain (8, CCND1_target_1- none (tonsil) c.920 − 5); 1C > T:splice(0.43, 225) EGFR_gain (13, EGFR_target_1- 29) Oropharynx posFBXW7_c.1099C > none none none (tonsil) T_p.R367* (0.22, 1380),KRAS_c.35G > A_p. G12D (0.25, 1892), PIK3CA_c.1633G > A_p.E545K (0.32,2759) Cell line pos none none none none Cell line pos noneNOTCH1:NM_017617: RICTOR_gain (7, RICTOR_target_1- none c.574G > T_p.39) G192* (0.96, 330) Cell line pos TP53_c.770T > G_p. none CCND1_gain(13, CCND1_target_1- none L257R (0.50, 787) 5) Cell line —CDKN2A_c.220G > none CCND1_gain (12, CCND1_target_1- none A_p.D74N(0.99, 144), 5); TP53_c.583A > T_p. MDM2_gain (8, MDM2_target_1- I195F(1.00, 1340) 11) Cell line neg none NOTCH1:NM_017617: CCND1_gain (12,CCND1_target_1- none c.158_158delA_p. 5); V53fs*47:frameshift EGFR_gain(16+, EGFR_target_1- (0.99, 181), 29) TP53:NM_000546: c.450_451insG_p.P153fs*28:frameshift (0.84, 351) — — none none none none — — none nonenone PTEN_loss (0, PTEN_target_1-9) — — none none none none — — nonenone BCL2L1_gain (9, BCL2L1_target_1- none 1) — — PIK3CA_c.3140A > nonenone none T_p.H1047L (0.25, 434) — — FBXW7_c.1099C > none none noneT_p.R367* (0.24, 940), KRAS_c.35G > A_p. G12D (0.23, 1288),PIK3CA_c.1633G > A_p.E545K (0.33, 1980) ¹The “column 3 somaticmutations” include non-synonymous, nonsense, conserved splice site,small indel, large deletion, or large amplification mutations. ²The“column 4 somatic mutations” are mutations that are not reported in thepublic databases or literature; they include a truncating mutation, suchas a nonsense, conserved splice site, frameshift small indel, or largedeletion) in known tumor suppressor genes.

Example 2: Methods

The following exemplifies certain embodiments of the methods andexperimental conditions used to identify the mutation described inTable 1. Additional screening can be done using, e.g., qRT-PCR analysisof cDNA prepared from a pre-selected tumor sample.

Massively parallel DNA sequencing was done on hybridization captured,adaptor ligation-based libraries using DNA isolated from archived fixedparaffin-embedded tissue. A combination of analysis tools were used toanalyze the data and assign DNA alteration calls.

Genomic DNA Sequencing

Sequencing of cancer genes was done using DNA from archived formalinfixed paraffin embedded (FFPE) tumor specimens from HNSCC patients.Sequencing libraries were constructed by the adapter ligation methodusing genomic DNA followed by hybridization selection with optimized RNAhybridization capture probes (Agilent SureSelect custom kit). Sequencingon the HiSeq2000 instrument (Illumina) was done using 49×49 paired readsto an average depth of 514×. Data processing and mutation assignmentsfor base substitutions, indels, copy number alterations and genomicrearrangements were done using a combination of tools optimized formutation calling from tumor tissue.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby the COSMIC database, available on the worldwide web atsanger.ac.uk/genetics/CGP/cosmic/; and the Institute for GenomicResearch (TIGR) on the world wide web at tigr.org and/or the NationalCenter for Biotechnology Information (NCBI) on the world wide web atncbi.nlm.nih.gov.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

1.-29. (canceled)
 30. A method of treating a subject with a cyclindependent kinase (CDK) inhibitor, wherein the subject has a squamouscell carcinoma of the head and neck (HNSCC), and wherein the CDKinhibitor inhibits one or both of cyclin dependent kinase 4 (CDK4) orcyclin dependent kinase 6 (CDK6), the method comprising the steps of:determining whether the subject has a mutation in a cell-cycle gene by:obtaining a biological sample from the subject; and performing an assayon the biological sample to determine if the subject has a mutation in acell-cycle gene; and if the subject has a mutation in a cell-cycle gene,then administering the CDK inhibitor to the subject.
 31. The method ofclaim 30, wherein the cell-cycle gene is chosen from a cyclin dependentkinase inhibitor 2A (CDKN2A) gene, a cyclin dependent kinase inhibitor2B (CDKN2B) gene, a Cyclin E1 (CCNE1) gene, a Cyclin D1 (CCND1) gene, aCyclin D2 (CCND2) gene, a Cyclin D3 (CCND3) gene, a CDK4 gene, a CDK6gene, or a gene described in Table 1 or Table
 4. 32. The method of claim30, wherein the subject has one or more of: (i) a loss-of-functionmutation in a CDKN2A gene; (ii) a gain-of-function mutation in a CCND1gene; (iii) a mutation or a mutant polypeptide described in Table 1 or4; or (iv) a mutant CDKN2A, CDKN2B, or CCND1 polypeptide.
 33. The methodof claim 30, wherein the mutation in the cell-cycle gene is detected ina nucleic acid molecule by one or more of: sequencing, a nucleic acidhybridization assay, an amplification-based assay, a PCR-RFLP assay,real-time PCR, screening analysis, FISH, spectral karyotyping, MFISH,comparative genomic hybridization, in situ hybridization, SSP, HPLC, ormass-spectrometric genotyping.
 34. The method of claim 30, wherein theCDK inhibitor is chosen from LEE011, LY-2835219, PD 0332991, Indisulam,AZD5438, SNS-032, SCH 727965, JNJ-7706621, indirubin, or Seliciclib. 35.The method of claim 30, further comprising administering a radiationtherapy to the subject, performing a surgery on the subject, or both.36. The method of claim 30, further comprising generating a personalizedcancer treatment report to memorialize the presence or absence of amutation in a cell-cycle gene in the subject.
 37. The method of claim30, wherein the biological sample is a blood sample, a serum sample, aurine sample, a tissue sample, or a buccal swab, or wherein thebiological sample comprises a cell from a tumor biopsy or a circulatingtumor cell.
 38. A method of treating a subject with a cyclin dependentkinase (CDK) inhibitor, wherein the subject has a squamous cellcarcinoma of the head and neck (HNSCC), the method comprising the stepsof: determining whether the subject is negative for the humanpapillomavirus (HPV−) or positive for the human papillomavirus (HPV+)by: obtaining a biological sample from the subject; and performing anassay on the biological sample to determine if the subject is HPV− orHPV+; and if the subject is HPV−, then administering the CDK inhibitorto the subject, and if the subject is HPV+, then administering ananti-cancer agent other than a CDK inhibitor to the subject.
 39. Themethod of claim 38, wherein the CDK inhibitor inhibits one or both ofcyclin dependent kinase 4 (CDK4) or cyclin dependent kinase 6 (CDK6).40. The method of claim 38, wherein the CDK inhibitor is chosen fromLEE011, LY-2835219, PD 0332991, Indisulam, AZD5438, SNS-032, SCH 727965,JNJ-7706621, indirubin, or Seliciclib.
 41. The method of claim 38,wherein the anti-cancer agent is chosen from4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), 5-azacytidine, 6-mercaptopurine,6-thioguanine, actinomycin D, amsacrine, bis-chloroethylnitrosurea,bleomycin, bryostatin-1, busulfan, carboplatin, chlorambucil, cisplatin,cetuximab, colchicine, cyclophosphamide, cytarabine, cytosinearabinoside, dacarbazine, daunorubicin, daunomycin, dactinomycin,deoxycoformycin, diethylstilbestrol (DES), doxorubicin, etoposide(VP-16), epirubicin, esorubicin, gemcitabine, hexamethylmelamine,hydroxyprogesterone, hydroxyurea, idarubicin, ifosfamide, irinotecan,mafosfamide, melphalan, methotrexate (MTX), methylcyclohexylnitrosurea,mithramycin, mitomycin C, mitoxantrone, nitrogen mustards, paclitaxel,pentamethylmelamine, prednisone, procarbazine, tamoxifen, taxol,teniposide, testosterone, trimetrexate, topotecan, vincristine, orvinblastine.
 42. The method of claim 38, further comprisingadministering an mTOR inhibitor, a PI3K inhibitor, a PI3K/mTORinhibitor, or a PI3K/Akt/mTOR inhibitor.
 43. The method of claim 42,wherein the mTOR inhibitor is rapamycin, a rapamycin derivative,resveratrol, or everolimus.
 44. The method of claim 42, wherein thePI3K/mTOR inhibitor is BEZ235, BGT226, BKM120, LY294002 or wortmannin.45. The method of claim 38, further comprising administering a radiationtherapy to the subject, performing a surgery on the subject, or both.46. The method of claim 38, wherein the subject has one or more of: (i)a loss-of-function mutation in a CDKN2A gene; (ii) a gain-of-functionmutation in a CCND1 gene; (iii) a mutation or a mutant polypeptidedescribed in Table 1 or 4; or (iv) a mutant CDKN2A, CDKN2B, or CCND1polypeptide.
 47. The method of claim 38, wherein the subject has amutation in a phosphoinositide-3-kinase, catalytic, alpha polypeptide(PIK3CA) gene; a phosphatase and tensin homolog (PTEN) gene; or aserine/threonine kinase 11 (STK11) gene.
 48. The method of claim 38,wherein the biological sample is a blood sample, a serum sample, a urinesample, a tissue sample, or a buccal swab, or comprises a cell from atumor biopsy or a circulating tumor cell.
 49. A method of treating asubject with a cyclin dependent kinase (CDK) inhibitor, wherein thesubject has a squamous cell carcinoma of the head and neck (HNSCC), themethod comprising the steps of: determining whether the subject ispositive for the human papillomavirus (HPV+) by: obtaining a biologicalsample from the subject; and performing an assay on the biologicalsample to determine if the subject is HPV+; and if the subject is HPV+,then administering an mTOR inhibitor, a PI3K inhibitor, a PI3K/mTORinhibitor, or a PI3K/Akt/mTOR inhibitor to the subject.
 50. The methodof claim 49, wherein the subject has a mutation in aphosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) gene; aphosphatase and tensin homolog (PTEN) gene; or a serine/threonine kinase11 (STK11) gene.
 51. The method of claim 49, wherein the mTOR inhibitoris rapamycin, a rapamycin derivative, resveratrol, or everolimus. 52.The method of claim 49, wherein the PI3K/mTOR inhibitor is BEZ235,BGT226, BKM120, LY294002 or wortmannin.
 53. The method of claim 49,further comprising administering a radiation therapy to the subject,performing a surgery on the subject, or both.
 54. The method of claim49, wherein the biological sample is a blood sample, a serum sample, aurine sample, a tissue sample, or a buccal swab, or comprises a cellfrom a tumor biopsy or a circulating tumor cell.